1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a potent inhibitor of keratinocyte proliferation, as well as a stimulator of epidermal terminal differentiation. In the present studies, we investigated the influence of epidermal growth factor (EGF) and insulin on the antiproliferative and differentiation activities of 1,25(OH)2D3. Our results indicate the following: (1) EGF caused a dramatic potentiation of the 1,25(OH)2 D3-induced inhibition of 3H-thymidine incorporation into keratinocytes in a dose-dependent manner; (2) insulin acted antagonistically on the EGF-dependent potentiation of the 1,25(OH)2D3-induced antiproliferative activity; (3) transforming growth factor-alpha potentiated 1,25(OH)2D3-induced antiproliferative activity similar to EGF; (4) the EGF effect was not dependent upon 1,25(OH)2D3 receptor mRNA up-regulation; and (5) removal of insulin from medium supplemented with growth factors significantly potentiated the 1,25(OH)2D3-induced inhibition on the number of basal cells and the 1,25(OH)2D3-dependent cornified envelope formation. In conclusion, the antiproliferative activity of 1,25(OH)2D3 in cultured normal human keratinocytes is greatly enhanced by EGF or transforming growth factor-alpha and reduced by insulin. Insulin also inhibits 1,25(OH)2D3-induced terminal differentiation of cultured normal human keratinocytes.