Stereochemical determinants of the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were investigated using the stereospecifically deuterated isotopomers (4R)-[4-2H1] NNK and (4S)-[4-2H1] NNK. Upon ip administration to groups of 20 female A/J mice, NNK and (4S)-[4-2H1]NNK exhibited similar lung tumorigenicity at three different doses, whereas (4R)-[4-2H1]NNK was 2-fold less tumorigenic at all three doses. In a parallel experiment, levels of O6-methylguanine and 7-methylguanine were 2-fold lower in lung DNA of mice treated with (4R)-[4-2H1]NNK than in mice treated with NNK or (4S)-[4-2H1]NNK. To corroborate these in vivo data, the in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda (Sf9)-expressed mouse cytochrome P450s 2A4 and 2A5. Kinetic isotope effects on the apparent Vmax (DV) for the product of NNK 4-hydroxylation, OPB, were 2.7 ± 0.2 and 2.8 ± 0.4 when (4R)- and (4S)-[4-2H1]NNK were incubated with mouse lung microsomes, respectively. The DV values for OPB formation were 3.2 ± 0.2 and 2.2 ± 0.2 when (4R)-[4-2H1]-NNK was the substrate for P450s 2A4 and 2A5, respectively, whereas they were 1.3 ± 0.1 and 1.1 ± 0.1 when (4S)-[4-2H1]NNK was the substrate for these respective enzymes. Analysis of an OPB derivative (10) for deuterium content by LC/MS confirmed the results from the kinetic assays and indicated that P450s 2A4 and 2A5 preferentially abstract the pro-R 4-hydrogen of NNK. The results obtained using Sf9-expressed P450s provide a rationale for the differences observed in the lung tumor and DNA adduct experiments, namely, that the attenuated tumorigenicity of (4R)-[4-2H1]NNK relative to (4S)-[4-2H1]NNK is due to prochiral selectivity during P450-catalyzed metabolic activation.