6-Nitrochrysene (NC) is a potent lung and liver carcinogen when administered in multiple doses to preweanling mice. We have investigated both the in vitro metabolism of (3H)NC by 9000 g supernatants (S9) prepared from the livers of preweanling mice and the in vivo metabolism of (3H)NC in these animals. The in vitro covalent binding of primary metabolites of NC to DNA after further reductive and/or oxidative metabolism was then examined in an attempt to define the metabolic activation pathway responsible for the formation of carcinogen-DNA adducts in NC-treated preweanling mice. NC-1,2-dihydrodiol, NC-9, 10-dihydrodiol, 6-aminochrysene (AC), and several unidentified compounds were found in ethyl acetate extracts of incubations containing (3H)NC and liver S9 from 1-or 8-day-old BLU: Ha mice. Comparison of the in vivo metabolism of NC in 1-day-old animals and 8-day-old animals which had been treated with NC on day 1 indicated that the formation of AC and the two NC dihydrodiols was greater in the younger animals. Further metabolism of NC-1, 2-dihydridiol by S9 from 8-day-old mice yielded AC-1,2-dihydrodiol as a major product. Incubation of AC-1,2-dihydrodiol, calf thymus DNA and liver microsomes from 3-methylcholanthrene-induced rats yielded a single major adduct that was chromatographically and chemically identical to the major adduct formed in (3HJNC-and (3H)-AC-treated preweanling mice. The results indicated that the major DNA adduct found in vivo is derived from the further metabolism of the proximate carcinogen AC-1, 2-dihydrodiol.