Benzidine diimine (BZDI), a reactive intermediate in the metabolic peroxidation of the carcinogen benzidine, has been reported to bind covalently to cellular proteins and nucleic acids. We have examined the nature of this interaction with DNA and have identified a major carcinogen-nucleoside adduct. BZDI (20-50 microM) reacted rapidly with DNA in vitro to give relatively high levels of covalently bound products (1-2 adducts/10(3) nucleotides; 30-45% yield). The binding was completely inhibited by addition of glutathione but was unaffected by acidic pH, air, free radical traps, or strong nucleophiles. Upon enzymatic hydrolysis of the BZDI-modified DNA and subsequent h.p.l.c., a major adduct was isolated and characterized by u.v., mass and proton magnetic resonance spectroscopy as N-(deoxyguanosin-8-yl)-benzidine. The identity of this adduct and its formation under various incubation conditions suggest a reaction mechanism that involves a simple deprotonation of the cationic diimine, formation of an electrophilic arylnitrenium ion, and covalent binding to guanine in the DNA.