3-Nitrobenzanthrone (3-NBA) is an urban air pollutant and rat lung carcinogen that is among the most potent mutagens yet tested in the Salmonella reversion assay. In the present study, 1 mg 3-NBA was administered orally to female F344 rats and DNA adduct formation was examined in liver, lung, kidney and five sections of the gastrointestinal (GI) tract at 6 hr, and 1, 2, 3, 5, and 10 days after administration. The DNA adduct patterns, analyzed by 32P-postlabelling followed by HPLC separation, were similar in all tissues and organs. Five of the adduct peaks cochromatographed with synthesized DNA adduct standards. Three of these unequivocally determined standards, dGp-C8-N-ABA, dGp-N2-C2-ABA, and dAp-N6-C2-ABA, were of the nonacetylated type, suggesting that at least part of the pathway for activation of 3-NBA proceeds through O-acetylation of the hydroxylamine intermediate. The two other DNA adduct standards, dGp-C8-C2-N-Ac-ABA, and dGp-N2-C2-N-Ac-ABA, were of the acetylated type, but there was some ambiguity in the characterization of these DNA adducts, since they varied inconsistently between samples and they also aligned with peaks found in controls. At 6 hr after treatment, the level of DNA adducts was highest in glandular stomach (relative adduct labeling (RAL), ˜70 adducts/108 normal nucleotides (NN)); adduct levels in this organ decreased at 24 hr, but increased afterwards. DNA adduct levels in the majority of organs were characterized by an early increase (from 6 hr to 3 days), which was followed by a decrease at 5 days and a maximum level 10 days after administration (RAL ˜120 adducts/108 NN for the lung, kidney and glandular stomach, ˜80 adducts/108 NN for the forestomach and ceacum, and ˜40 adducts/108 NN for the liver, small intestine, and colon). This pattern was consistent with pathological observations during autopsy showing high levels of tissue damage in the GI tract; the tissue damage included hemorrhages, loss of villous surface structure in the small intestine, as well as intestine fragility and oedema of the adipose tissue around the GI-tract. Tissue damage decreased and DNA adduct levels increased at 10 days after administration. These observations suggest that 3-NBA not only exerts acute toxic effects, but that the bioavailability is affected by storage in tissues and later becomes available, resulting in the increased DNA adduct levels at the later time points of collection.