The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) can be activated metabolically by cytochrome(s) P450 to DNA-damaging agents that result in the formation of tumors in various organs of several animal models. In the present study, 30-min incubations at 37 degrees containing 5 mg/mL pooled human placental microsomes, 36 nmol NNK (including 2 microCi [5-3H]NNK) and a 5 mM concentration of either NADH, NADPH, or both cofactors together resulted in the formation of 11.43 +/- 0.32, 35.40 +/- 4.64, and 44.05 +/- 1.66 pmol 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)/mg protein/min (mean +/- SD, N = 3), respectively. Similar experiments using 7, 9, and 11 mM NADH, NADPH, and both cofactors together in equimolar concentrations yielded results that suggest that NADH- and NADPH-dependent reductions of NNK are catalyzed by different enzymes. Computer simulations for the production of NNAL based on various kinetic models corroborated the conclusion drawn from the empirically derived data. In human placental microsomes, the Km,app and Vmax,app for the formation of NNAL were 1021.9 +/- 251.5 microM and 4360.7 +/- 991.7 pmol/mg protein/min, respectively. Inhibition of cytochrome P450-dependent activities by carbon monoxide and dicumarol (100 and 200 microM) resulted in an average increase of NNAL production of 40 and 56%, respectively, suggesting that P450-dependent biotransformation of NNK is occurring in the absence of inhibitors. Similarly, polyclonal goat IgG against rabbit P450 reductase resulted in a 12% increase in the production of NNAL when compared with control values. Thirty micromolar rutin, ethacrynic acid, cibacron blue 3GA, and iodoacetic acid, known inhibitors of certain human carbonyl reductase(s), incubated with placental microsomes containing an equimolar concentration of NNK, did not have a significant effect on the production of NNAL. These results establish that: (1) cytochromes P450 are likely involved in the metabolism of NNK by human placental microsomes, (2) metabolism of NNK to NNAL by human placental microsomes is catalyzed by an NADPH-dependent carbonyl reductase(s) and an NADH-dependent carbonyl reductase(s), and (3) reduction of NNK to NNAL is catalyzed by a placental microsomal carbonyl reductase(s) not previously described.