Recent studies have indicated a lack of correlation between hepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and the carcinogenicity of peroxisome proliferators (PP) and suggested that DNA in intact hepatic nuclei may be insensitive to increases in 8-OHdG resulting from PP exposure. The possibility that PP-induced elevations in acyl CoA oxidase (ACO) activity might result in oxidative damage to mitochondrial DNA (mtDNA) was therefore investigated by feeding male F344 rats the hepatocarcinogenic PP Wy-14,643 (Wy, 0.1% in the diet) for 3, 6, 11, or 22 weeks, or clofibric acid (CA, 0.5% in the diet) for 22 weeks. Following the respective PP exposures, hepatic peroxisomal acyl CoA oxidase activity was determined and DNA isolated from either mitochondria or unfractionated liver homogenates and analysed for the presence of 8-OHdG. PP treatment caused an increase in ACO activity (10- to 15-fold) at all time points examined and an increase of 8-OHdG (1.5- to 2-fold) in DNA isolated from unfractionated liver homogenates following PP treatment for 11 or 22 weeks. No increase of 8-OHdG in mtDNA was detected. However, quantitation of a PCR amplified region from the D-loop of mtDNA demonstrated a 2- to 3-fold increase in the relative amount of mtDNA in DNA isolated from unfractionated liver homogenates following 3, 11, and 22 weeks exposure to Wy or CA (22 weeks only). In addition, a slight increase in the mitochondrial volume density (1.4-fold) was observed in electron micrographs of liver samples from rats exposed to Wy for 22 weeks. These results (i) demonstrate that PP treatment, at levels which cause an increase in ACO activity, does not cause oxidative damage to mtDNA, and (ii) suggest that one reason for the observed increase of 8-OHdG in DNA from unfractionated liver homogenates may be an increase in the amount of mtDNA present in these samples. Furthermore, these studies provide additional evidence against a role of oxidative DNA damage, measured as 8-OHdG, in PP-induced rodent hepatocarcinogenesis and suggest that alterations in mitochondria or other effects may be more pertinent to PP-related carcinogenesis.