Fatty acyl-CoA oxidase null mice (AOX−/−) develop hepatocellular carcinomas in 100% of animals between 10 and 15 months. We evaluated spontaneously developed HCC in AOX−/− mice for PPARα, PPARα regulated genes and peroxisome volume density and compared with adjacent non-neoplastic liver and liver in wild-type (AOX+/+) and heterozygous (AOX+/−) mice. The level of PPARα mRNA was 2.5-fold higher in HCC compared to the adjacent liver. mRNAs of PPARα regulated genes such as peroxisomal bifunctional enzyme, thiolase, cytochrome P450 CYP4A1 and CYP4A3 were similar in HCC and adjacent liver and increased by 7-to 22-fold compared with wild-type and heterozygous mice. Immunoblot analysis of HCC showed high amounts of PPARα, peroxisomal bifunctional enzyme and thiolase. Electron microscopic examination revealed 3.8 and 8.3-fold increase in the volume density of peroxisomes in HCC and adjacent liver, respectively, compared to the volume density in wild-type mice. These results demonstrate that spontaneously developed HCC in AOX−/− mice display a similar type of pleiotropic responses to high levels of PPARα ligands as the non-neoplastic liver. The changes observed in HCC and adjacent liver in AOX−/− mice were identical to those observed in rats and mice exposed to peroxisome proliferators.