We investigated the role of N-acetyltransferases (NAT) in prostate cancer (PCa) susceptibility. NAT are polymorphic in the population and metabolize important carcinogenic products directly involved in the tumor initiation process. This prospective case-control study utilized the polymerase chain reaction-based restriction fragment length polymorphism method and comprised a cohort of consecutive 478 individuals: 126 men with prostate cancer; 101 men with benign prostatic hyperplasia (BPH); and a control health population of 177 female and 74 male blood donors from the same region. NAT2 slow or fast acetylators genotypes were determined by the combination of four variant alleles. Lifetime occupational history, dietary patterns, cigarette smoking and other anamnestic data were obtained by interviews. We were not able to find any correlation among smoking, dietary patterns, parameters of tumor aggressiveness or patient outcome and any NAT2 genotypes or phenotypes considered in separate or in different combinations. However, there was an association between NAT2T481C (OR=0.47; 95% CI=0.26-0.84; P=0.01) and NAT2A803G (OR=0.57; 95% CI=0.33-0.97; P=0.04) polymorphisms and PCa protection. Conversely, the presence of NAT2G857A genotype increased the risk of PCa more than 3 times (OR=3.57; 95% CI=1.39-9.15; P=0.005). Slow acetylator NAT2*7A and NAT2*6B genotypes occurred in 10.31% of PCa but in none of BPH patients (P=0.0007). The control health population confirmed the results and allowed the exclusion of possible biases caused by gender influence on genotype inheritance and by the inclusion of not diagnosed prostate diseases patients among the control individuals. We suggest that the investigation of germline polymorphisms of NAT2 gene may be useful in the assessment of Latin American patients at risk of BPH and PCa.