The role of liver plasma membrane (LPM) fluidity in the pathogenesis of intrahepatic cholestasis in rats was assessed by comparing the effects of ethinyl estradiol, a cholestatic agent, and spironolactone on membrane fluidity and bile flow. Spironolactone is a steroid that has some feminizing actions but that lacks the phenolic A ring necessary for estrogens to cause cholestasis. Bile flow was reduced 42% (p less than 0.01) by ethinyl estradiol and increased 22% (p less than 0.05) by spironolactone; however, both agents produced a significant reduction of membrane Na+, K+-ATPase activity (p less than 0.01) and fluidity (p less than 0.01). The decreased fluidity persisted in liposomes prepared from the total lipid extract as well as the phospholipid extract of these membranes. Both agents produced similar significant increases in the cholesterol ester content and cholesterol-to-phospholipid molar ratio of the membranes. In addition, ethinyl estradiol and spironolactone increased the membrane sphingomyelin content (15% and 11%, respectively); however, neither agent altered the fatty acid composition of the phospholipids. Because the decreased fluidity persisted in liposomes prepared from phospholipids extracted from the LPMs of treated rats, changes in membrane cholesterol are not the sole cause of the altered membrane fluidity. Rather, the increased sphingomyelin is at least partially responsible for these changes. Also, because ethinyl estradiol and spironolactone produce similar changes in LPM lipid composition and fluidity but disparate effects on bile flow, membrane fluidity as assessed by fluorescence polarization does not appear to be the rate-limiting determinant of bile flow in estrogen-induced cholestasis.