Trichoderma harzianum strain T22 parasitizes and controls many phytopatogenic fungi and is applied commercially as biological control agent. The production of hydrolitic enzymes appears to be a key factor in the parasitic process. We tested the endo-esochitinolitic and glucanolitic activities of culture filtrates of T22 grown under carbon and nitrogen starvation or in presence of biomass or cell walls of the phytopathogenic fungi Botrytis cinerea, Rhizoctonia solani and Pythium ultimum. The highest level of enzimatic activities was found in culture where the mycoparasite interacted with a phytopathogenic fungus. Therefore we used a proteomic approach to investigate changes in the complex mixture of extracellular proteins secreted by T. harzianum strain T22 in order to identify proteins of potential biotechnology value for commercial and industrial use.Proteome technology has greatly enhanced our ability to conduct functional genomics studies. Nevertheless only a few studies have been published so far on the fungal extracellular proteome. Sample preparation remains the most critical step in analyses based on two-dimensional gel electrophoresis (2-DE), and it requires to be optimized for each specific application. In this study, our first aim was to set up the extraction protocol of the extracellular proteins secreted by T . harzianum strain T22 when it was grown in vitro .The secreted proteins were analysed by two-dimensional electrophoresis (2-DE) and substantial changes in the extracellular proteome of the mycoparasite have been observed. Comparing the 2D maps of the fungus grown in minimal medium with glycerol as carbon source (used as control condition) with those obtained in inducing conditions, a lot of novel proteins appeared. The higher number of novel and up-regulated spots was obtained in the presence of Rhizoctonia solani biomass. Other spots were specifically up-regulated by the interaction with different plant pathogens. Differentially expressed proteins were subjected to matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and N-terminal Edman sequencing. The in silico analyses of some of the novel and up-regulated spots showed interesting homology to hypothetical and putative proteins from other fungal species. These include novel enzymes, such as glycosylhydrolases and metalloprotease, proteins with conserved domains involved in pathogen-host interactions, such as Ras that regulates signal transduction pathways or LRRs that is involved in host recognition, etc. Work is in progress to demonstrate the role of some of these proteins in biocontrol and ability to induce systemic resistance. Trichoderma harzianum strain T22 parasitizes and controls many phytopatogenic fungi and is applied commercially as biological control agent. The production of hydrolitic enzymes appears to be a key factor in the parasitic process. We tested the endo-esochitinolitic and glucanolitic activities of culture filtrates of T22 grown under carbon and nitrogen starvation or in presence of biomass or cell walls of the phytopathogenic fungi Botrytis cinerea, Rhizoctonia solani and Pythium ultimum. The highest level of enzimatic activities found in culture where the mycoparasite interacted with a phytopathogenic fungus. Therefore we used a proteomic approach to investigate changes in the complex mixture of extracellular proteins secreted by T. harzianum strain T22 in order to identify proteins of potential biotechnology value for commercial and industrial use .Proteom technology has greatly enhanced our ability to conduct functional genomics studies. Synchron Only a few studies have been published so far on the fungal extracellular proteome. Sample preparation remains the most critical step in analyses based on two-dimensional gel electrophoresis (2-DE), and it requires to be optimized for each specific application. In this study, our first aim was to set up the extraction protocol of the extracellular proteins secreted by T. harzianum strain T22 when it was grown in vitro. The secreted proteins were analysed by two-dimensional electrophoresis (2-DE) and substantial changes in the extracellular proteome of the mycoparasite have been observed. Comparing the 2D maps of the fungus grown in minimal medium with glycerol as carbon source (used as control condition) with those obtained in inducing conditions, a lot of novel proteins appeared. The higher number of novel and up-regulated spots was obtained in the presence of Rhizoctonia solani biomass. Other spots were specifically up-regulated by the interaction with different plant pathogens. Differentially expressed proteins were subjected to matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and N-terminal Edman sequencing. The in silico analyzes of some of the novel and up-regulatedAskely interesting homology to hypothetical and putative proteins from other fungal species. These include novel enzymes, such as glycosylhydrolases and metalloprotease, proteins with conserved domains involved in pathogen-host interactions, such as Ras that regulates signal transduction pathways or LRRs that is involved in host recognition, etc. Work is in progress to demonstrate the role of some of these proteins in biocontrol and ability to induce systemic resistance.