The synthetic estrogens ethinyl estradiol (EE) and mestranol (M) are weak complete hepatic carcinogens and potent tumor promoters. In vivo, EE and M cause a rapid but transient increase in liver growth. However, studies in cultured female rat hepatocytes indicate that EE is not a strong complete hepatic mitogen but rather enhances epidermal growth factor (EGF)-induced DNA synthesis and is thus classified as a co-mitogen (Yager, J.D., Zurlo, J. and Ni, N. (1991) Proc. Soc. Exptl. Biol. Med., 198, 667-674). The endogenous estrogen 17 beta-estradiol (E2) also exhibits co-mitogenic activity, enhancing the fraction of hepatocytes undergoing DNA synthesis induced by both EGF and transforming growth factor alpha (TGF-alpha) (Ni, N. and Yager, J.D. (1994) Hepatology, 19, 183-192). The objectives of the study reported here were: (1) to determine whether the co-mitogenic effects of EE and E2 extend to other synthetic estrogens including mestranol and diethylstilbestrol, and to alpha-zearalanol, a natural product with estrogenic activity; (2) to compare the co-mitogenic effects of endogenous estrogens including E2, estrone, estriol and the catechol metabolites 2- and 4-hydroxy-estradiol; and (3) to determine whether the conditioned medium from E2-treated hepatocytes has co-mitogenic activity. Female rat hepatocytes in primary culture were exposed to the various estrogens +/- TGF-alpha and DNA synthesis was determined by measuring [3H]thymidine incorporation into extracted DNA. The results show that the co-mitogenic effects previously observed with EE and E2 also extend to all of these estrogens and to the E2 catechol metabolites. Although the co-mitogenic potency of these estrogens does not correlate with their reported affinities to the estrogen receptor, their estrogenicity appears necessary since the non-estrogenic metabolite 2-methoxy-estradiol lacks co-mitogenic activity. In addition, enhancement of TGF-alpha-induced DNA synthesis by conditioned medium from E2-treated cells supports the notion that a metabolite mediates its co-mitogenic effect.