Polycyclic aromatic hydrocarbons (PAHs) are a diverse group of widespread environmental pollutants, some of which have been found to be estrogenic or antiestrogenic. Recent data have shown that hydroxylated PAH metabolites may be responsible for the estrogenic effects of some PAHs. The purpose of this study was to investigate the effects of several PAHs, as well as their monohydroxylated metabolites, on estrogen receptors (ERs), ERα and ERβ. Three parent PAHs and their monohydroxylated metabolites were each evaluated using transcriptional reporter assays in isogenic stable cell lines to measure receptor activation, competitive binding assays to determine ligand binding, and bioluminescence resonance energy transfer assays to assess dimerization. Finally, the estrogenic effects of the hydroxylated metabolites were confirmed by quantitative real-time PCR of estrogen-responsive target genes. Although the parent PAHs did not induce ERα or ERβ transcriptional activity, all of the monohydroxylated PAHs (1-OH naphthanol, 9-OH phenanthrene, 1-OH pyrene) selectively induced ERβ transcriptional activity at the concentrations tested, while not activating ERα. Additionally, the monohydroxylated PAHs were able to competively bind ERβ, induce ERβ homodimers, and regulate ERβ target genes. Although monohydroxylated PAHs appeared to have weak agonist activity to ERβ, our results showed that they can elicit a biologically active response from ERβ in human breast cancer cells and potentially interfere with ERβ signaling pathways.