Urinary metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides, termed total NNAL, have recently been shown to be good predictors of lung cancer risk, years before diagnosis. We sought to determine the contribution of several genetic polymorphisms to total NNAL output and inter-individual variability. The study subjects were derived from the Harvard/Massachusetts General Hospital Lung cancer case-control study. We analyzed 87 self-described smokers (35 lung cancer cases and 52 controls), with urine samples collected at time of diagnosis (1992–1996). We tested 82 tagging SNPs in 16 genes related to the metabolism of NNK to total NNAL. Using weighted case status least squares regression, we tested for the association of each SNP with square-root (sqrt) transformed total NNAL (pmol per mg creatinine), controlling for age, sex, sqrt packyears and sqrt nicotine (ng per mg creatinine). After a sqrt transformation, nicotine significantly predicted a 0.018 (0.014, 0.023) pmol/mg creatinine unit increase in total NNAL for every ng/mg creatinine increase in nicotine atp< 10E-16. ThreeHSD11B1SNPs andAKR1C4rs7083869 were significantly associated with decreasing total NNAL levels:HSD11B1rs2235543 (p= 4.84E-08) and rs3753519 (p= 0.0017) passed multiple testing adjustment at FDRq= 1.13E-05 and 0.07 respectively,AKR1C4rs7083869 (p= 0.019) did not, FDRq= 0.51. HSD11B1 and AKR1C4 enzymes are carbonyl reductases directly involved in the single step reduction of NNK to NNAL. TheHSD11B1SNPs may be correlated with the functional variant rs13306401 and theAKR1C4SNP is correlated with the enzyme activity reducing variant rs17134592, L311V.