Aromatic amines constitute one of the most extensively studied classes of chemical carcinogens. Although monocyclic aromatic amines are generally regarded as weak carcinogens, a recent epidemiologic study of bladder cancer found that the arylamine 3,5-dimethylaniline (3,5-DMA) may play a significant role in the etiology of this disease in man. Investigations using experimental animals also strongly suggested that DNA adducts-of indeterminate structure-formed by 3,5-DMA might account for its presumptive activity. The present study was undertaken to determine the structures of the major DNA adducts formed in vitro by the known, and possibly carcinogenic, N-hydroxylated metabolite. Calf thymus DNA (ct-DNA) was modified by reaction with N-acetoxy-3,5-dimethylaniline (N-AcO-3,5-DMA). After enzymatic hydrolysis of DNA to individual 2'-deoxyribonucleosides, adduct profiles were determined using HPLC/MS. 3,5-DMA formed four major DNA adducts, one to 2'-deoxyguanosine (dG), two to 2'-deoxyadenosine (dA), and one to 2'-deoxycytidine (dC). Reactions of N-AcO-3,5-DMA with dG, dA, and dC produced the same adducts as reaction with ct-DNA with very similar profiles. Adducts were isolated chromatographically and unambiguously characterized as N-(deoxyguanosin-8-yl)-3,5-dimethylaniline (dG-C8-3,5-DMA), 4-(deoxyadenosin- N(6)-yl)-3,5-dimethylaniline (dA- N(6)-3,5-DMA), N-(deoxyadenosin-8-yl)-3,5-dimethylaniline (dA-C8-3,5-DMA), and N-(deoxycytidin-5-yl)-3,5-dimethylaniline (dC-C5-3,5-DMA) by high-resolution mass spectra (HR-MS) and NMR spectroscopy including (1)H NMR, (13)C NMR, and two-dimensional NMR. This report includes the first detailed description of a dC adduct of an aromatic amine. The present results provide chemical support for a carcinogenic mechanism of action by 3,5-DMA based on N-hydroxylation and the intermediacy of a nitrenium ion in the formation of DNA adducts.