4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, has been reported to induce lung adenomas in A/J mice. The initial step in NNK-induced lung tumorigenesis is thought to be the formation of methylated DNA bases, which can lead to DNA mutations. Among methylated DNA bases, O⁶-methylguanine (O⁶MeG) is recognized as the major promutagenic DNA adduct, inducing primarily GC to AT transitional mutations. In this study the stability and recovery of O⁶MeG during sample preparation and quantification was monitored and optimized. We then quantified the concentrations of O⁶MeG in various organs over the first 24 h following acute treatment with a fixed dose of NNK (Study I) and the dose-response relationship in selected organs 4 h after treatment (Study II). In Study I, mature female A/J mice were administered NNK (10 µmole/mouse, i.p.) and were sacrificed 0, 2, 4, 8, 12, or 24 h after treatment. In Study II, mice received NNK at doses of 0, 2.5, 5, 7.5, or 10 µmole/mouse and were sacrificed 4 h after treatment. DNA from selected organs was extracted, acid-hydrolyzed, and analyzed for O⁶MeG using HPLC with UV and fluorescence detection. O⁶MeG was detected in the lung and liver of all the mice and in the kidney of 67% of the mice receiving NNK treatment, but was absent in brain and heart. The concentration of O⁶MeG in the lung and liver reached its maximum within 2 h after NNK treatment and remained stable throughout the next 24 h. Four hours after administration of NNK at doses of 0 to 10 µmole/mouse, the concentration of O⁶MeG in lung and liver demonstrated a dose-response relationship. At the dose of 10 µmole NNK/mouse, the O⁶MeG concentrations averaged over the initial 24 h in liver, lung, and kidney were 108, 13, and 20 pmole/µmole of guanine, respectively. These results indicate that the presence of O⁶MeG DNA adducts in NNK treatment does not correlate with the tumorigenesis, since lung is the only target organ for NNK-induced tumorigenesis in A/J mice, but DNA adducts were present in much higher concentrations in liver. Thus, factors other than the initial concentration or repair of O⁶MeG DNA adducts are critical in the tumorigenesis of NNK in A/J mice.