Estradiol inhibits cardiac fibroblast growth and may protect against cardiac remodeling associated with heart disease. However, the mechanisms by which estradiol attenuates cardiac fibroblast growth remain unclear. Because cardiac fibroblasts express cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT) capable of converting estradiol to hydroxyestradiols and methoxyestradiols, respectively, and because hydroxyestradiols and methoxyestradiols (estradiol metabolites with little affinity for estrogen receptors) are potent inhibitors of cardiac fibroblast growth, we hypothesized that the antimitogenic effects of estradiol are mediated via hydroxyestradiols and/or methoxyestradiols. The inhibitory effects of estradiol (1 to 100 nmol/L) on serum-stimulated (3)H-thymidine incorporation (DNA synthesis), (3)H-proline incorporation (collagen synthesis), and cell number (proliferation) were enhanced (P<0.005) by CYP450 inducers 3-methylcholanthrene (10 micromol/L) and phenobarbital (10 micromol/L). Moreover, the inhibitory effects of estradiol were blocked by the CYP450 inhibitor 1-aminobenzotriazole (10 micromol/L) and the COMT inhibitors quercetin (10 micromol/L) and OR486 (10 micromol/L). In contrast to estradiol, the modulators of CYP450 and COMT were poor ligands for estrogen receptors (binding affinity less-than-or-equal 0.0001% versus estradiol). In cardiac fibroblasts, both quercetin and OR486 inhibited the metabolism of hydroxyestradiol to methoxyestradiol and blocked the inhibitory effects of hydroxyestradiol on cardiac fibroblast proliferation and DNA and collagen synthesis. The abrogating effects of quercetin and OR486 on the metabolism and antimitogenic effects of 2-hydroxyestradiol were mimicked by 20 micromol/L norepinephrine and isoproterenol, substrates for COMT. Our findings provide evidence that estradiol can inhibit cardiac fibroblast growth via an estrogen receptor--independent pathway that involves the local metabolism of estradiol to methoxyestradiols.