Materials and Methods: Limbal biopsies were taken from normal human limbus (Central Eye Bank of Iran), and were cultured as an explant on amniotic membrane and plastic dish. Cells were exposed to the air after 14 days of culture. The expression of proposed limbal stem cells markers (p63, ABCG2), corneal markers (K3/12, Connexin43) and also HSPs (90, hsc70) were analyzed by RT-PCR, immunocytochemistry and flowcytometry pre and post air exposure at day 14 (before air exposure) and 30 (after air exposure) respectively.Results: ABCG2 positive cells with high expression of p63 and low expression of K3/K12 and Connexin43 were considered as limbal stem cells while ABCG2 negative cells with low expression of p63 and high expression of K3/K12, Connexin43 were assumed as corneal cells. Our data showed that HSPs expressed strongly at mRNA level before and after air exposure while they expressed in different pattern at protein level. It was interested that HSP90 proteins were expressed weakly in limbal stem cells at day 14 on both groups, but its expression increased after air exposure at day 30 (p<0.05) only in AM group on the other hand HSC70 were expressed strongly on plastic dish in compared with amniotic membrane (p<0.05) before air exposure but after air exposure its expression has significant increasing in AM group.Conclusion: We assumed hsp90 has important role during differentiation process but only in early stage of differentiation because its increasing was in AM group,and in this group we had TAC cells(early differentiated cells) after air exposure but Hsc70 expression may related with differentiation process in both early and terminally stage of differentiation in cultured limbal stem cell.