The influence of ethinyl estradiol (EE) on the effects of dietary cholesterol on the biliary saturation index and on the rate-limiting hepatic enzymes of cholesterol synthesis, hydroxymethylglutaryl-CoA-reductase, and bile acid synthesis, 7 alpha-hydroxylase, were determined. Four groups of 12 male hamsters were treated for 1 month with EE, 15 micrograms per kg per day, or placebo vehicle administered intraperitoneally and fed either a standard diet, 0.8 mg of cholesterol per g of food, or high cholesterol diet, 2.4 mg of cholesterol per g. The high cholesterol diet increased the saturation index to 1.00 +/- 0.03 (P less than 0.01) from 0.65 +/- 0.02 in untreated hamsters on the standard diet. EE treatment on the high cholesterol diet further increased (P less than 0.01) the saturation index to 1.15 +/- 0.02. The high cholesterol diet decreased (P less than 0.01) hydroxymethylglutaryl-CoA-reductase activity from 308 +/- 16 pmoles per mg per min in untreated hamsters on the standard diet. The addition of EE treatment had no effect on hydroxymethylglutaryl-CoA-reductase activity. The high cholesterol diet increased (P less than 0.01) 7 alpha-hydroxylase activity from 23 +/- 1.0 pmoles per mg per min in untreated hamsters on the standard diet. The addition of EE decreased (P less than 0.01) 7 alpha-hydroxylase activity from that in untreated hamsters on the standard diet. The conclusions are as follows: (1) EE prevented dietary cholesterol-induced stimulation of cholesterol 7 alpha-hydroxylase activity; (2) EE enhanced the ability of dietary cholesterol to induce saturated bile; and (3) gallstone formation in estrogen-treated women may result from impaired metabolism of dietary cholesterol.