Mutagenic oxidative DNA base damage increases with age in prostatic tissue. Various factors may influence this increase including: increased production of reactive oxygen species, increased susceptibility to oxidative stress, alterations in detoxifying enzyme levels or defects in DNA repair. Using liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry, we show increased levels of oxidative DNA base lesions, 8-hydroxyguanine (8-oxoG), 8-hydroxyadenine (8-oxoA) and 5-hydroxycytosine (5OHC) over the baseline in PC-3 and DU-145 prostate cancer cells following exposure to ionizing radiation and a repair period. Nuclear extracts from PC-3 and DU-145 prostate cancer cell lines are defective in the incision of 8-oxoG, 5OHC and thymine glycol (TG) relative to the non-malignant prostate cell line. Consistent with reduced expression of OGG1 2a, incision of 8-oxoG is reduced in PC-3 and DU-145 mitochondrial extracts. We also show a correlation between severely defective incision of TG and 5OHC and reduced levels of NTH1 in PC-3 mitochondria. The antioxidant enzymes, glutathione peroxidase (GPx), catalase and superoxide dismutases (SOD1, SOD2), have altered expression patterns in these cancer cell lines. Genetic analysis of the OGG1 gene reveals that both PC-3 and DU-145 cell lines harbor polymorphisms associated with a higher susceptibility to certain cancers. These data suggest that the malignant phenotype in PC-3 and DU-145 cell lines may be associated with defects in base excision repair and alterations in expression of antioxidant enzymes.