Introduction: Shigella is a facultative intracellular pathogen that uses the host actin cytoskeleton protein for intra- and intercellular spread. The aim of this study was to determine the distribution of icsA gene and IcsA expressed protein bands among Shigella flexneri strains isolated from 3 clinical centers in Tehran. Material and Methods: Two hundred and seventy five isolated Shigella flexneri strains were identified by standard microbiological and biochemical methods. DNA isolation was performed using sodium perchlorate method. Hot start-PCR was done with 2 pairs of primers and the products were separated through agarose gel (0.8%) in TAE buffer. DNA fragments were visualized by ethidium bromide staining under UV illumination. Whole membrane preparation was used to examine the protein profiles and identification of probable IcsA (120-kda) protein band by SDS-PAGE. Results: From 100 isolated Shigella flexneri strains, both bands of 1600 bp and 1709 bp were detected in 46 isolates (46%). A 120 kDa band which seems to be related to IcsA protein was detected in 46 isolates (46%). The protein bands varied between 30 and 150 kDa. Discussion: IcsA is both necessary and sufficient for actin assembly in Shigella flexneri. Since icsA gene and IcsA protein band were not found in all Shigella strains, it seems that not all strains have the same pathogenesis. On the other hand, since the demonstration of icsA gene by PCR in all Shigella strains (46%) corresponded to the presence of a 120 kDa protein band by SDS-PAGE (46%), it seems that both tests may confirm each other. However, the PCR may be more accurate than SDS-PAGE.