The cytostatic drug dacarbazine [DTIC, 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide] is strongly carcinogenic in rats. Bioactivation of DTIC yields a methylating intermediate but the extent of interaction with cellular macromolecules has not previously been reported. Following a single i.p. injection of [14C-methyl]DTIC, exhalation of 14CO2 occurred with a t1/2 max of approximately 2 hr (0.95 mg/kg) and 2.5 hr (95 mg/kg). Of the total radioactivity administered, 8.5% was exhaled as 14CO2; 54% was excreted via the urine, predominantly as unchanged DTIC. In liver, kidney and lung, formation of 7-[14C]methylguanine in DNA and RNA was directly proportional with dose. DNA methylation by a single dose of DTIC (9.8 mg/kg; 5 hr survival time) was highest in liver (35 mumoles 7-methylguanine/mole guanine), followed by kidney (25 mumoles) and lung (20 mumoles). The remainder tissues showed 7-methylguanine concentrations approximately 50% of those in liver DNA, with the exception of the brain which had a very low extent of DNA modification (approximately 1 mumole/mole guanine). At the specific radioactivity used (48 mCi/mmole), the promutagenic base O6-methylguanine was only detectable in liver, kidney, lung, and stomach DNA (0.6-0.8 mumoles/mole guanine). Autoradiographic studies revealed a diffuse distribution of reaction products in rat liver. In contrast, N-nitrosodimethylamine and related carcinogens known to be bioactivated by the hepatic cytochrome P-450 system show a predominantly centrilobular distribution. This difference may be due to the greater stability of proximate carcinogens generated by alpha-C hydroxylation at one of the methyl groups of DTIC.