The estrogenic activities of bisphenol A (BPA) and its major metabolite BPA glucuronide (BPA-G) were assessed in a number of in vitro and in vivo assays. BPA competed with [3H]-17beta-estradiol (E2) for binding to mouse uterine cytosol ER, a glutathione S-transferase (GST)-human ER D, E, and F domain fusion protein (GST-hERalphadef) and full-length recombinant hERbeta. The IC(50) values for E2 were similar for all three receptor preparations, whereas BPA competed more effectively for binding to hERbeta (0.96 microM) than to either mouse uterine cytosol ER (26 microM) or GST-hERalphadef (36 microM). In contrast, BPA-G did not competitively displace [3H]E2 from any of the ER preparations. In MCF-7 cells transiently transfected with Gal4-hERalphadef or Gal4-hERbetadef, BPA induced reporter gene activity with comparable EC(50) values (71 and 39 microM, respectively). No significant induction of reporter gene activity was seen for BPA-G. Cotreatment studies showed that concentrations of (10 microM) BPA and BPA-G did not antagonize E2-induced luciferase mediated through either Gal4-hERalphadef or Gal4-hERbetadef. In vivo, the uterotropic effect of gavage or subcutaneous (sc) administration of 0.002-800 mg of BPA/kg of body weight/day for three consecutive days was examined in immature rats. Dose-related estrogenic effects on the rat uterus were observed at oral doses of 200 and 800 mg/kg and at sc doses of 10, 100, and 800 mg/kg. These results demonstrate that BPA competes more effectively for binding to ERbeta, but induces ERalpha- and ERbeta-mediated gene expression with comparable efficacy. In contrast, BPA-G did not exhibit any in vitro estrogenic activity. In addition, there was a clear route dependency on the ability of BPA to induce estrogenic responses in vivo.