4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice following a single intraperitoneal (i.p.) injection. NNK-induced lung tumorigenesis is thought to involve O⁶-methylguanine (O⁶MeG) formation, leading to GC à AT transitional mutations in mouse lung genomic DNA. Formation of O⁶MeG requires metabolic activation of NNK, primarily through a-hydroxylation by cytochrome P₄₅₀ enzymes. Nicotine, cotinine (the major metabolite of nicotine), and aqueous cigarette tar extract (ACTE) have all been shown to effectively inhibit metabolic activation of NNK to its mutagenic form, most likely due to competitive inhibition of cytochrome P₄₅₀ enzymes. The objective of the current study was to monitor the effects of cotinine and cigarette smoke (CS) on the molecular dosimetry of O⁶MeG in target tissues of mice during the acute phase of NNK treatment. To test the effect of cotinine, mature female A/J mice received a single intraperitoneal injection of NNK (0, 2.5, 5, 7.5 and 10 µmole/mouse) with cotinine administered at 50 µmole/mouse in 3 separate i.p. injections, i.e. , 30 min. before, immediately after, and 30 min. after NNK treatment. To test the effect of whole smoke exposure on NNK-related O⁶MeG formation, mice were exposed to smoke generated from Kentucky 1R4F reference cigarettes at 0, 0.4, 0.6 or 0.8 mg wet total particulate matter/liter (WTPM/L) for 2hrs. with a single i.p. injection of NNK (0, 3.75 or 7.5 µmole/mouse) midway through the exposure. Cigarette smoke alone failed to yield detectable levels of O⁶MeG. NNK-induced O⁶MeG formation in both lung and liver was significantly (p<0.05) reduced by cotinine and by cigarette smoke exposure. Our results demonstrate that NNK-induced DNA adducts in A/J mice are dramatically reduced when NNK is administered together with either cotinine, the major metabolite of nicotine, or the parental complex mixture, cigarette smoke.