Benzidine, a human urinary bladder carcinogen, induces hepatic tumors in mice and rats. In this study, [3H]benzidine was administered in drinking water to mice for 1 week, and the covalent binding of the carcinogen to hepatic DNA was then determined. A single carcinogen:DNA adduct was detected which decreased in concentration by approximately 50% at 1 day after treatment and then remained at a nearly constant level for at least 7 days. Injection of radiolabeled benzidine or N-acetyl-benzidine into rats also resulted in a single carcinogen:DNA adduct that was chromatographically identical to that obtained in mouse liver. While administration of benzidine and N-acetylbenzidine resulted in high levels of the adduct in rat hepatic DNA, injection of N,N'-[ring-14C]diacetylbenzidine did not give detectable binding (less than 0.3 residue/mg DNA). The same carcinogen:DNA adduct found in rat and mouse liver was prepared synthetically by: (a) hydrolysis of calf thymus DNA reacted with N-hydroxy-N'-acetylbenzidine at pH 5; and (b) reaction of N-acetoxy-N,N'-diacetylbenzidine with deoxyguanosine and subsequent selective deacetylation of the product with methanolic ammonia. The in vitro and in vivo products were found to have identical high-pressure liquid chromatography retention times and to exhibit similar pH-dependent solvent partitioning characteristics. Mass and nuclear magnetic resonance spectral data on the synthetic products established the structure of the hepatic adduct as N-(deoxyguanosin-8-yl)-N'-acetylbenzidine. The structural isomer, N-(deoxyguanosin-8-yl)-N-acetylbenzidine, was synthesized by treatment of N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine (the intermediate in b) with carboxylesterase and was shown to be chromatographically distinct from the in vivo adduct. Similarly, the nonacetylated derivative, N-(deoxyguanosin-8-yl) benzidine, was synthesized by carboxylesterase treatment of N-(deoxyguanosin-8-yl)-N'-acetylbenzidine and was shown not to occur in rat and mouse liver DNA. These data indicate that the metabolic activation of benzidine to an ultimate carcinogen in rats and mice does not involve N-hydroxybenzidine or sulfotransferase-catalyzed activation of N-hydroxy-N,N'-diacetylbenzidine. The remaining pathways for metabolic conversion of benzidine to an ultimate carcinogenic species are discussed in relation to liver and urinary bladder carcinogenesis.