Cigarette smoking is the predominant risk factor for bladder cancer (BC). Major carcinogens present in tobacco smoke include a number of aromatic and heterocyclic amines. Two distinct N-acetyl transferase (NAT) enzymes, NAT1 and NAT2, play important roles in the bio-activation and detoxification of these carcinogens. Genes encoding NAT1 and NAT2 are highly polymorphic among human populations, and these polymorphisms result in rapid or slow acetylator phenotypes. Recent studies have suggested that variant alleles leading to slow acetylation by the NAT2 enzyme or rapid acetylation by the NAT1 enzyme constitute possible risk factors for bladder cancer. In this case-control study, we sought to determine whether NAT1 and NAT2 polymorphisms are associated with bladder cancer risk in the largest sample size to date. PCR-RFLP assay was used to determine the presence of NAT1 and NAT2 polymorphisms in 507 Caucasian BC patients and 513 age-, gender-, and ethnicity-matched healthy controls. Overall, we found no significant association between BC risk and NAT1 NAT1*10 allele (OR=0.95; 95% CI 0.73-1.25). However, our data suggested that NAT2 slow acetylator genotypes were associated with a significant increased risk of BC (OR=1.31; 95% CI, 1.01-1.70). This elevated risk appeared more evident in older individuals (OR=1.41; 95% CI, 1.01-1.98) than in younger individuals (OR=1.15; 95% CI, 0.76-1.74). Moreover, the risk was greater for heavy smokers (OR=2.11; 95% CI, 1.33-3.35) than light smokers (OR=0.96; 95% CI, 0.61-1.53) and never smokers (OR=1.23; 95% CI, 0.79-1.90). Finally, a joint effect between NAT2 slow acetylators and heavy smokers was observed. Using never smokers with NAT2 rapid acetylator genotypes as a reference group, heavy smokers with NAT2 slow acetylator genotypes showed an over six-fold increase in BC risk. In a multiplicative interaction model, the interaction term was statistically significant (P=0.02). Our data suggest that having a NAT2 slow acetylator genotype is a significant risk factor for BC, particularly in smokers and older individuals.