The incubation of [3H]1,8-dinitropyrene with Salmonella typhimurium TA98NR followed by isolation of the DNA from these cells, hydrolysis of the DNA to nucleosides, butanol extraction of the hydrolysate and purification by reversed-phase liquid chromatography afforded a single product. Calf thymus DNA, after treatment with N-hydroxyl-1-amino-8-nitro-pyrene, was hydrolyzed, extracted and purified in a similar fashion to give a single compound which was shown to be the deoxyguanosine derivative 1-N-(2'-deoxyguanosin-8-yl)-amino-8-nitropyrene by a combination of proton n.m.r. and u.v.-vis. spectroscopy and fast atom bombardment mass spectrometry. The DNA adducts formed in vivo and in vitro exhibited identical chromatographic and chemical behavior. Under acidic or basic conditions in the vivo and in vitro adducts were converted to identical products. Reduction of the adduct gave a new, highly fluorescent product that had a fluorescence emission spectrum identical to that of 1,8-diaminopyrene.