The effects of ethynylestradiol on endogenous bile acids, their capacity to conjugate and excrete intravenously infused cholic acid, the concentrations of biliary cholesterol and lecithin, and the individual molecular species of phosphatidylcholine have been determined in male and female Sprague-Dawley rats. Endogenous biliary bile acids were analyzed by gas-liquid chromatography-mass spectrometry. Eleven bile acids were identified and several minor bile acids, primarily muricholates, could not be completely characterized. After 5 days of treatment with ethynylestradiol (1 mg/kg per day), the percentage of cholic acid decreased and the percentage of 6beta-hydroxylated bile acids, including several monounsaturated species, increased. Ethynylestradiol caused a decrease in bile acid-independent bile flow. Intravenous infusion of cholic acid at a high concentration caused cholestasis in control animals but, after ethynylestradiol treatment, cholestasis developed during the infusion of a much lower concentration of cholate, indicating a lowered threshhold for bile acid-induced cholestasis. In the treated rats, there was a slight increase in excretion of unconjugated endogenous bile acids, and a striking impairment of conjugation of intravenously administered cholic acid. One of the few sex-related differences observed was an increased concentration of biliary phospholipids in untreated male rats. Both phospholipid and cholesterol concentrations in the bile were higher in the treated animals. The molar percentage of cholesterol was always 1-2%, but it was slightly higher in treated animals, especially males. Ethynylestradiol treatment also affected biliary phospholipid by causing a marked increase of phosphatidylcholine species containing palmitic and oleic acid residues and a decrease of species containing stearic and linoleic acid residues. There was no increase in biliary excretion of long chain polyunsaturated species, which might have indicated damage to membranes, in response to ethynylestradiol either alone or with cholic acid infusion. Some of these ethynylestradiol-induced changes in biliary bile acid and lipid excretion are probably peculiar to the rat, but others, such as the increase in molar percentage of cholesterol and cholestasis, may be relevant to disorders in man, especially cholesterol gallstones and idiopathic cholestasis of pregnancy.