Aim: To identify and characterize a novel gene with potential roles in testis development and spermatogenesis.Methods: A cDNA microarray was constructed from a human testis large insert cDNA library and hybridized with probes of human or mouse adult and fetal testes. Differentially expressed genes were isolated and sequenced. RT-PCR was used to test the tissue distribution of the genes of interest and in situ hybridization was performed to localize the gene expression in the mouse testis. A range of bioinformatical programs including Gene Runner, SMART, NCBI Blast and Emboss CpGPlot were used to characterize the new gene\'s feature. Results: A novel testis-specific gene,NYD-SP5, was differentially expressed in fetal and adult testes. The deduced protein structure of NYD-SP5 was found to contain an IQ motif (a short calmodulin-binding motif containing conserved Ile and Gln residues), a Carbamate kinase-like domain, a Zn-dependent exopeptidase domain and a lactate dehydrogenase (LDH) C-terminal-like domain. RT-PCR analysis revealed that NYD-SP5 was predominantly expressed in the testis but not in other 15 tissues examined. In situ hybridization and RT-PCR examinations revealed that the expression of NYD-SP5 was confined in the male germ cell but not present in the somatic cell in the testes. Conclusion: NYD-SP5 is a newly found testisspecific gene with potential roles in testis development and spermatogenesis through a calmodulin-activated enzyme.