The ability of benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon, to bind to the alpha and beta forms of the estrogen receptor (ER) and to produce in vitro and in vivo estrogenic responses was assessed. While B[a]P did not significantly compete with tritiated 17beta-estradiol ([3H]E2) for binding to human ERalpha (bacterially expressed fusion protein consisting of GST and the DEF domains of the receptor) or human ERbeta (full-length) in vitro, four hydroxylated B[a]P metabolites (1-, 3-, 7-, and 9-hydroxy-B[a]P) were able to bind appreciably to both isoforms at concentrations of up to 10 mM, with all metabolites showing a stronger binding preference for hERbeta than for hERalpha. In transiently transfected MCF-7 human breast cancer cells, B[a]P and 3-hydroxy- and 9-hydroxy-B[a]P were similarly potent in causing human ERalpha- and mouse ERbeta-mediated reporter gene expression (EC50 values 0.3-2 mM), while B[a]P and the 1- and 7-hydroxy-B[a]P metabolites were more active in the mERbeta system. Three daily doses as high as 10 mg/kg oral B[a]P or 20 mg/kg subcutaneous 3- or 9-hydroxy-B[a]P did not significantly increase uterine wet weight or lactoferrin mRNA expression in C57BL/6 or DBA/2 mice. These results suggest that the alpha and beta isoforms of the ER have a different capacity to bind and interact with B[a]P and its major hydroxylated metabolites in vitro, however, these compounds did not cause increased uterine weight or lactoferrin expression in the mouse.