Cimetidine, a histamine H2 antagonist, is known to interfere with the metabolism of exogenous drugs by binding to cytochrome P450. We examined the possibility that cimetidine might also inhibit the cytochrome P450-dependent biotransformation of endogenous compounds such as steroid hormones. Utilizing a radiometric assay and normal male volunteers, the acute effect of intravenous cimetidine (300 mg loading dose followed by 50 mg/hr) was determined. The extent of 2-hydroxylation of estradiol was reduced by 25% from 29.6 +/- 4.4% (mean +/- SEM) before, to 22.9 +/- 4.0% during cimetidine infusion (n = 8; p less than 0.0005). Following oral cimetidine (800 mg b.i.d.) for 2 wk, estradiol 2-hydroxylation was decreased by 40% from 31.7 +/- 2.3% to 19.7 +/- 2.4% (n = 9; p less than 0.0001) but 16 alpha-hydroxylation of estradiol was unaffected. Concomitantly, the urinary excretion of 2-hydroxyestrone was decreased by 25% (p less than 0.002) and the serum estradiol concentration was increased by 20% (p less than 0.04). In contrast, ranitidine, a second generation H2 receptor antagonist, had no effect on estradiol hydroxylation following 150 mg b.i.d. for 2 wk. The inhibition of estradiol 2-hydroxylation and the increase in serum estradiol concentrations caused by cimetidine administration may help to account for the symptoms of hyperestrogenization reported in long-term cimetidine therapy.