The hepatic lobular localization of alcohol dehydrogenase was determined in male, female and castrated male rats. Alcohol dehydrogenase immunoreactive protein and activity were increased in female and castrated rats as compared to normal male rats. By immunohistochemistry, alcohol dehydrogenase protein was found localized principally in the perivenous area of the hepatic lobule in all of the animals. Alcohol dehydrogenase activity eluted from the male rat liver, during bidirectional digitonin perfusion, exhibited a pattern characteristic of cytosolic enzymes predominantly localized to the perivenous area. The elution of the enzyme was more rapid during cava-porta than porta-cava perfusion occurring in close association with the elution of glucokinase, an enzyme localized principally in the perivenous area. By contrast, elution of lactate dehydrogenase, which is located predominantly in the periportal area, preceded elution of alcohol dehydrogenase during porta-cava perfusion, but followed it during cava-porta perfusion. These differences were less apparent in the cava-porta than in the porta-cava direction. The predominant localization of alcohol dehydrogenase immunoreactive protein and activity to the perivenous area of hepatic lobule was not affected by sexual difference or increase in the enzyme following castration.