The ability of benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon, to bind to the a and b 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 17b-estradiol ([³H]E2) for binding to the human ERa (bacterially expressed fusion protein consisting of GST and the DEF domains of the receptor) or human ERb (full-length) in vitro, two hydroxylated B[a]P metabolites showed strong differential binding to the a and b receptor isoforms. 3-hydroxy- and 9-hydroxy-B[a]P competed fully with [³H]E2 for binding to hERb, with IC₅₀ values of 49 nM and 1.0 mM respectively. However with hERa, concentrations of up to 10 mM caused only 60% and 30% displacement of [³H]E2 respectively. In transiently transfected MCF-7 human breast cancer cells, B[a]P, 3-hydroxy- and 9-hydroxy-B[a]P were similarly potent in causing human ERa- and mouse ERb-mediated reporter gene expression (EC₅₀ values 0.3 - 2 mM). Furthermore, at a concentration of 10 mM all three compounds in both the hERa and mERb systems induced reporter gene expression to levels equal to that of E2 at 10 nM, with one exception: B[a]P in the hERa system induced levels only 25% of those of E2. 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 a and b 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 do not cause increased uterine weight or lactoferrin expression in the mouse.