To compare the extent of modulation by various growth factors on clonal growth and differentiation between corneal and stem cell-containing limbal epithelium. A reported serum-free clonal growth assay was used. The mitogenic response was measured by colony forming efficiency, colony size, and bromodeoxyuridine labelling index; the differentiation was assessed by AE-5 monoclonal antibody staining. As compared to controls in an insulin-containing basic medium, the addition of epidermal growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, and a high dose of nerve growth factor was mitogenic for both epithelia. Cholera toxin was also mitogenic, but platelet-derived growth factor might not have, and insulin-like growth factor type I lacked an additive mitogenic effect with insulin. The mitogenic effect of epidermal growth factor differed from the others in its dose-dependent down-regulation and in having more migratory cells in epidermal growth factor-stimulated colonies. In contrast to these mitogens, transforming growth factor-beta 1 exhibited a clear inhibitory effect in the controls as well as in epidermal growth factor- or fibroblast growth factor-stimulated cultures. Transforming growth factor-beta 1's inhibitory effect was correlated with strong AE-5 staining, whereas the mitogenic effect of epidermal growth factors, fibroblast growth factors or nerve growth factors was correlated with the presence of negative AE-5 colonies. There exist potential dynamic interactions among various growth-modifying cytokines in controlling epithelial growth and differentiation. The lack of specific activation of limbal stem cells in this culture assay also suggests that other unidentified factor(s) might be regulating stem cell functions.