Male rats were fed a cholesterol-free diet for 5 weeks, followed by a 2% cholesterol diet for 4 weeks. Another group of rats was continuously fed a cholesterol-free diet. A third group was fed standard pelllets during the whole experiment. Hepatic microsomal protein and cholesterol contents and drug-metabolizing enzyme activities were measured. The cholesterol-rich diet increased microsomal protein content and this increase disappeared after trypsin digestion of microsomal membranes. Microsomal cholesterol content was enhanced three-fold by cholesterol feeding. Cytochrome P-450 concentration, NADPH cytochrome c reductase and aryl hydrocarbon hydroxylase activities showed only minor changes following cholesterol feeding. The p-nitroanisole O-demethylase and ethoxycoumarin deethylase activities were doubled by cholesterol in comparison to cholesterol-free diet. Trypsin digestion activated the UDP-glucuronosyltransferase enzyme eight- to ten-fold on a protein basis. Trypsin treatment increased the cholesterol activation of UDP-glucuronosyltransferase when compared to the activity in native microsomes. The data suggest that dietary cholesterol regulates the cholesterol content of microsomal membranes. The activities of drug-metabolizing enzymes are also altered, possibly due to the compositional changes of the membranes.