Xenobiotic metabolizing enzymes are involved in the detoxification of many carcinogens and may be important in modulating cancer susceptibility. CYP1A1, CYP2E1, GSTM3, and NAT2 polymorphisms were determined in peripheral blood DNA of 231 oral cancer patients and 212 hospital controls in Rio de Janeiro, Brazil, using the PCR–RFLP technique. NAT2 polymorphism distribution was different between cases and controls (P = 0.035), with an overrepresentation of NAT2*11 mutant allele in controls. Risk analysis showed that NAT2 4/4 individuals (OR = 1.95, 95% CI = 1.05–3.60) and combined GSTM3 and NAT2 heterozygotes (OR = 1.94, 95% CI = 1.04–3.66) were at increased oral cancer risk. No statistically significant association was observed for CYP1A1 and CYP2E1 polymorphisms. Our results suggest that NAT2 polymorphism, alone or combined with GSTM3, may modulate susceptibility to oral cancer in Rio de Janeiro.