Mouse mammary epithelial cells cultured on collagen gels multiplied and produced casein and alpha-lactalbumin in response to insulin, cortisol, and PRL. The addition of epidermal growth factor (EGF) at 50 ng/ml increased the total number of epithelial cells by 30-40% and thymidine incorporation into DNA 4.7-fold after 5 days of culture. In contrast, EGF inhibited hormonal induction of the synthesis of casein and alpha-lactalbumin in those cells by about 45% and 55%, respectively, without inhibiting total protein synthesis. Furthermore, EGF decreased casein mRNA activity by 55% and increased total mRNA activity by 66% in cells cultured with the three hormones. These effects of EGF were apparent at 0.1 ng/ml and were maximal at 50-100 ng/ml and could be reversed by its removal from the medium, followed by the addition of anti-EGF antibody. The inhibition of casein synthesis by EGF was unaffected by the concentrations of insulin, cortisol, and PRL. Other growth factors, such as fibroblast growth factor, multiplication-stimulating activity, nerve growth factor, and platelet-derived growth factor, did not simulate the effects of EGF. Cytarabine (1 microgram/ml), which inhibited thymidine incorporation into DNA by 94%, did not block the inhibitory action of EGF on casein synthesis. These results suggest that EGF serves as a regulator of hormone-dependent growth and differentiation of mammary epithelial cells.