Cytochrome P450 (CYP) 2A5 is involved in the metabolism of carcinogens like aflatoxin B1 and N-nitrosodiethylamine (NDEA), and CYP2A5 levels are increased in some pathological states of the liver (e.g., infectious hepatitis and porphyria). We analyzed the expression of CYP2A5 during experimental liver carcinogenesis in three different mouse strains (C3H/He, C57BL/6J, and B6C3F1) with immunohistochemical techniques and in situ hybridization. In normal liver, CYP2A5 protein and mRNA were detected in centrilobular hepatocytes only. Phenobarbital treatment increased the number of CYP2A5-positive centrilobular hepatocytes and the CYP2A5-positive areas were extended into the middle zone in all strains, but periportal hepatocytes remained negative. Fifty percent of the spontaneous foci in untreated mice, over 90% of the foci in mice treated with NDEA or phenobarbital and all of the hepatocellular adenomas and carcinomas displayed positive immunostaining and a strong CYP2A5 mRNA signal by in situ hybridization. In the liver tumors metastasized to the lung, expression of CYP2A5 had largely disappeared. CYP2A5 expression in neoplastic and putative preneoplastic lesions, although sometimes heterogeneous, was apparently independent of the typical zonal expression pattern in normal tissue. As expected, the C57BL/6J mice developed fewer foci and tumors than the C3H/He and B6C3F1 mice, but the phenotype of CYP2A5 overexpression was similar in all the strains. Our data suggest that the increased expression of CYP2A5 may play an important role in the development of liver cancer in mice and may be used as a novel marker for spontaneous and NDEA-induced mouse liver foci.