The mycotoxin ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rodents. However, the mechanism of OTA-induced tumor formation is unknown and conflicting results have been obtained regarding the potential of OTA to bind to DNA. OTA is poorly metabolized, and no reactive intermediates capable of interacting with DNA have been detected in vitro or in vivo. Recently, a hydroquinone/quinone redox couple and a carbon-bonded OTA-deoxyguanosine (OTA-dG) adduct formed by electrochemical oxidation and photoreaction of OTA have been reported and suggested to be involved in OTA carcinogenicity. This study was designed to characterize the role of DNA binding and to determine if formation of these derivatives occurs in vivo and in relevant activation systems in vitro using specific and sensitive methods. Horseradish peroxidase activation of OTA and its dechlorinated analogue ochratoxin B (OTB) yielded ochratoxin A-hydroquinone (OTHQ), but the postulated OTA-dG adduct was not detectable using LC-MS/MS. In support of this, no OTA-related DNA adducts were observed by 32P-postlabeling. In vivo, only traces of OTHQ were found in the urine of male F344 rats treated with high doses of OTA (2 mg/kg body wt) for 2 weeks, suggesting that this metabolite is not formed to a relevant extent. In agreement with the in vitro data, OTA-dG was not detected by LC-MS/MS in liver and kidney DNA extracted from treated animals. In addition, DNA binding of OTA and OTB was assessed in male rats given a single dose of 14C-OTA or 14C-OTB using accelerator mass spectrometry, a highly sensitive method for quantifying extremely low concentrations of radiocarbon. The 14C content in liver and kidney DNA from treated animals was not significantly different from controls, indicating that OTA does not form covalent DNA adducts in high yields. In summary, the results presented here demonstrate that DNA binding of OTA is not detectable with sensitive analytical methods and is unlikely to represent a mechanism for OTA-induced tumor formation.