The relative roles of hepatitis B virus (HBV) and aflatoxin and their possible mechanism of interaction in the etiopathogenesis of hepatocellular carcinoma (HCC) are not understood. One hypothesis is that viral infection and associated liver injury alter expression of carcinogen-metabolizing enzymes. We tested this hypothesis in an HBV-transgenic mouse model in which a synergistic interaction occurs between aflatoxin B1 (AFB1) and HBV in the induction of HCC (Sell et al., Cancer Res 51:1278-1285, 1991). In this transgenic mouse lineage, overproduction of the HBV large envelope protein results in progressive liver cell injury, inflammation, and regenerative hyperplasia. Initially, two cytochrome P450s of importance in AFB1 metabolism in the mice were identified, namely Cyp2a-5 and Cyp3a, using specific antibodies and chemical inhibitors. The expression of these P450 isoenzymes and an alpha-class glutathione S-transferase (GST) isoenzyme, YaYa, were examined. Increased expression and altered distribution of Cyp2a-5 were demonstrated, by immunohistochemical analysis, to be associated with the development of liver injury in mice and to increase with age between 1 and 12 months. Cyp3a expression was also increased in HBV-transgenic mice, but the increase was not as clearly related to age. GST YaYa levels were the same in HBV-transgenic mice and their nontransgenic littermates of all ages. These results show that expression of specific cytochrome P450s is altered in association with overexpression of HBV large envelope protein and liver injury in this model. This may have general relevance to human HCC, the etiology of which is associated with a diverse range of liver-damaging agents.