This study was designed to investigate the feasibility of using a serum-free and feeder layer-free culture system on serial expansion of rabbit limbal and corneal epithelial cells by evaluating the cell morphology, growth kinetics and gene expression pattern. Isolated epithelial cells(EC) from limbal and corneal regions of the rabbit corneoscleral tissues were culture expanded until three passages following initial culture in serum-free medium(SFM) supplemented with human corneal growth supplement(HCGS). EC morphology was examined daily with cell count and viability recorded at each passage by trypan blue exclusion test. Growth rate, number of cell doublings and total cell yield were measured and compared in each passage. The expression of Integrin β1; putative epidermal stem cell marker; Cytokeratin 12 and Connexin 43; differentiation markers for corneal epithelium; were detected by semiquantitative reverse transcriptase-polymerase chain reaction(RT-PCR). Limbal EC was able to retain compact and small cells morphology throughout the culture period. Corneal EC demonstrated a varying morphology with abundance of large, squamous cells and showed senescence earlier in culture. Limbal EC exhibited a greater proliferative activity demonstrated by higher growth rate, cumulative cell doublings and total cell yield compared to corneal EC. Integrin β1 expression was detected in freshly isolated and cultured limbal EC in all passages with relatively strong expression noted at the initial passage (P0). Corneal EC showed a very weak expression of Integrin β1 throughout the culture period. Cytokeratin 12 and connexin 43 were expressed in both freshly isolated limbal and corneal EC. Both corneal differentiation markers were expressed in cultured limbal and corneal EC in serial passages. These results suggested that corneal epithelium can be serially expanded in serum-free and feeder layer-free culture system with limbal EC showing a greater proliferative capacity while retaining its stemness via putative epidermal stem cell marker; Integrin β1 expression.