The basis for gender-dependent differences in rates of glucuronidation of xenobiotics is uncertain. To clarify this issue, the glucuronidation of p-nitrophenol was compared in liver microsomes from adult male and female rats. The activity of native UDP-glucuronosyltransferase was 47% higher in microsomes from male than from female rats. Immunoblotting of microsomal protein with anti-UDP-glucuronosyltransferase antiserum revealed 66% more immunoreactive protein in male microsomes. A kinetic method for measuring glucuronidating enzyme content confirmed the result of the immunoblot. Responses of UDP-glucuronosyltransferase to activation by palmitoyllysophosphatidylcholine or high pressure indicated that the activity of the enzyme was more latent in male than in female microsomes. Differences in enzyme latency could be due to differences in membrane structure. A comparison of microsomal fatty acid composition revealed significantly higher levels of oleic and linoleic acids and lower levels of stearic and docosahexaenoic acids in male than in female microsomes. The phospholipid composition, ratio of cholesterol:phospholipid, and membrane fluidity were similar in male and female microsomes. These results indicate that gender-dependent differences in UDP-glucuronosyltransferase activity are due to differences in both the amount and functional state of the enzyme.