An in vivo expression technology (IVET) was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of these, two were identified as alkylation-related genes; one was related to metabolism; one encoded a transcriptional regulator; three were identified as insertion elements; three ap- peared to be antisense to genes involved in the transmethylation,biosyntheseis, and phos- photransferase system;and three were predicted to encode polypeptides with unknown functions. Intracellular survival assavs showed that the mutants of alkA,citC and wcaJ genes had lower capability of intracellular replication or survival than the the wild-type strain.The results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements. However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. Very possibly, yaiC gene was involved in the other mechanism of S. flexneri virulence. This study might lead to a better understanding of the intra- cellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of three were identified as insertion elements; three ap- peared to be antisense to genes involved in the transmethylation, biosyntheseis, and phos-photransferase system; and three were predicted to encode polypeptides with unknown functions. Intracellular survival assavs showed that the mutants of alkA, citC and wcaJ genes had lower capability of intracellular replication or survival than the wild-type strain. The results indicate that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of int However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. However, yaiC gene was involved in the other virology. flexneri virulence. This study might lead to a better understanding of the intra-cellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.