To study the proliferation and differentiation of limbal stem cells (LCs) in vitro and examine the effects of epithelial growth factor (EGF) on cell proliferation.The cells were cultured in DMEM/F12 medium. The proliferation and differentiation of cultured cells were studied by colony-forming efficiency (CFE), immunohistochemistry staining and Western blot examination.LCs could be passaged 5 times in this culture condition. The CFE of stem cells was (5.07 +/- 2.35)%, while that of the central epithelial cells (CCs) was (1.12 +/- 0.86)%. The rates of freezing and recovery were (0.43 +/- 0.22)% in LCs and (0.16 +/- 0.07)% in CCs. Limbal stem cells could be stimulated to proliferate better by EGF at the concentration of 5 and 20 ng/ml. The staining of keratin 3 (K3) to stem cells in the primary culture was negative, but positive in the second and third passages. However, the expression of K3 by Western blot examination was positive in the primary and second passages but negative in the fourth one.It is better for limbal stem cells to be used for transplantation and drug screening in the primary culture.