Administration of the synthetic estrogen ethinyl estradiol (17alpha-ethinyl-1,3,5-estratriene-3,17beta-diol) decreases hepatic Na(+),K(+)-ATPase (ATP phosphohydrolase; EC 3.6.1.3) activity and bile flow to 50% and alters the composition and structure of surface membrane lipid in rats. Although the content of phospholipids was not changed by treatment, free cholesterol (130%) and cholesterol esters (400%) were increased in liver surface membrane fractions. These observations correlate with changes in membrane viscosity, as shown by electron spin resonance probes. Both rotational correlation time, using the isotropic probe methyl (12-nitroxyl)stearate, and the order parameter, determined by the anisotropic probe 5-nitroxylstearic acid, were significantly increased in liver surface membrane fractions from rats treated with ethinyl estradiol. Administration of Triton WR-1339, a nonionic detergent that corrects hepatic and serum lipid changes caused by ethinyl estradiol treatment, restored toward normal elevated membrane lipids and viscosity as well as Na(+),K(+)-ATPase activity and bile flow. Although restoration of normal liver surface membrane structure and function may be due to reversal of abnormal lipid composition, detergents also may directly alter membrane enzyme activity. Addition of Triton WR-1339 in vitro increased Na(+),K(+)-ATPase activity and reduced membrane viscosity of surface membranes from rats treated with ethinyl estradiol. Triton had no effect on either parameter in normal membrane preparations. Studies of membrane structure and function both in vivo and in vitro suggest that alterations in lipid composition may alter Na(+),K(+)-ATPase function and bile flow.