Perivenular hepatocytes are the first cells within the liver lobule to display signs of toxicity following long‐term alcohol use. In an attempt to explain this phenomenon, we have examined the hepatic intralobular distribution in rats and man of P450IIE1, a P‐450 isozyme that not only oxidizes ethanol but is also inducible by this agent. Frozen liver sections and microsomes were prepared from male Sprague‐Dawley rats pair‐fed liquid diets containing 36% of total calories as either ethanol or carbohydrate (control) for 10 to 21 days. Frozen sections or microsomes were also prepared from liver biopsy samples obtained from 17 male patients with diverse drinking histories. Immunohistochemical staining was performed using the peroxidase‐antiper‐oxidase method after liver sections were reacted with monospecific antibody (IgG) directed against human P450IIE1. Immunoreaction intensity was blindly rated in order to provide a semiquantitative assessment of P450IIE1 levels in perivenular, midzonal and periportal hepatocytes. At low applied anti‐P450IIE1 IgG concentrations (2.5 μg per ml), P450IIE1 immunostaining was observed exclusively within the perivenular area in sections from all ethanol‐treated rats, whereas no visible immunoreaction was found in sections from their pairfed controls. At higher applied antibody concentrations (15 μg per ml), panlobular antigen immunostaining was observed in five of the six ethanol‐treated animals, and P450IIE1 could now also be detected in perivenular hepatocytes from the control rats. In accordance with these immunohistochemical findings, protein blotting with anti‐P450IIE1 IgG revealed a 7.5‐fold increase in liver microsomal P450IIE1 content in ethanol‐treated animals when compared to their pair‐fed controls. With human liver, perivenular P450IIE1 immunostaining was observed only in biopsy sections obtained from recently drinking alcoholics (abstinence period of 1 day) when limiting concentrations (5 μg per ml) of the primary antibody were used. Increasing the applied anti‐P450IIE1 IgG concentration to 15 μg per ml resulted in perivenular staining of the immunogen in liver sections from abstinent alcoholics (abstinence period of 4 to 8 days) and nondrinkers as well. Immunoblot analysis of human liver microsomes disclosed that the hepatic microsomal P450IIE1 content in recently drinking alcoholics was 4‐fold higher than that found in nondrinkers. Our results show that, in both rats and in man, P450IIE1 is normally localized within the perivenular region, or zone 3, of the liver lobule, and that induction of P‐450IIE1 by prolonged alcohol consumption occurs primarily within the same acinar region. Since P450IIE1‐catalyzed ethanol oxidation results in the formation of acetaldehyde, an established cytotoxin, and free radicals capable of peroxidizing cell membranes, the elevated P450IIE1 levels found in perivenular hepatocytes after chronic alcohol intake suggest that increased production of ethanol‐derived toxic metabolites occurs in zone 3 cells, which, in turn, may explain their enhanced susceptibility to alcohol‐mediated damage.