The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in mice and rats and is a putative human lung carcinogen. NNK undergoes cytochrome P450-mediated metabolic activation to DNA-binding intermediates but is also extensively reduced to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in vivo. Because NNAL is also tumorigenic, the carcinogenicity of NNK may actually be governed by the metabolic activation of NNAL, rather than direct activation of NNK. Metabolism of NNK and NNAL at the 4-position generates the same critical DNA lesion, O6-methylguanine, the levels of which are correlated to tumorigenicity in the A/J mouse model. In an effort to better understand the bioactivation of NNAL and the effect of carbinol-carbon stereochemistry on prochiral selectivity at the 4-position, (R)- and (S)-NNAL, along with the stereospecifically 4-deuterated diastereomers (1R,4R)-[4-2H1]NNAL, (1R,4S)-[4-2H1]NNAL, (1S,4R)-[4-2H1]NNAL, and (1S,4S)-[4-2H1]NNAL, were synthesized. The in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda-expressed mouse cytochrome P450 2A5. Carbinol-carbon stereochemistry did not appreciably influence stereo-selectivity at the 4-position in the metabolism of these compounds by mouse lung microsomes or P450 2A5 but did influence the regiochemistry of metabolism. The ratio of 4- to N-methyl hydroxylation was approximately 1:1 for the A/J mouse lung microsome-mediated metabolism of all substrates, but this ratio was higher for (1S) substrates than for their (1R) counterparts when P450 2A5 was used. Interestingly, P450 2A5 converted substrates with (1S) stereochemistry to the respective N-oxides, but this metabolite was not formed from substrates with (1R) stereochemistry. Furthermore, P450 2A5 catalyzed the formation of NNK from (1S) substrates at significantly greater maximal rates than from (1R) substrates. The implications of these differences in metabolism for the tumorigenic mechanism of NNAL are discussed.