The arylamine N-acetyltransferase (NAT2) is a polymorphic enzyme which is expressed in the liver in a genotype-determined manner. NAT2 is involved in activation and inactivation of carcinogens through N-acetylation. We studied the role of this polymorphism in the development of hepatocellular carcinoma (HCC). One hundred consecutive patients diagnosed for HCC and 258 healthy volunteers were studied for NAT2 genotype. The occurrence of seven enzyme-inactivating and silent point mutations in the coding region of the NAT2 gene was studied by mutation-specific PCR amplification. An excess of subjects homozygous for NAT2 loss of function alleles was observed among patients with HCC (68% vs 53.9% controls). The relationship between the slow acetylator NAT2 genotype and HCC risk is more pronounced in patients lacking serum HBV and HCV markers. The additional determination of alleles of the cytochrome P450 2D6 (CYP2D6) gene in the same subjects confirmed our previous findings that subjects with two active CYP2D6 genes are at increased risk of developing HCC. The genetic polymorphism of NAT2 is a relevant factor in the risk for developing HCC (inverse odds ratio slow vs rapid = 1.8; 95% CI 1.1-3.0). The inverse odds ratio for subjects with two risk genotypes (two defect NAT2 genes and two or more active CYP2D6 genes) is 2.6 (95% CI 1.6-4.4) for all patients with HCC, and 5.6 (95% CI 1.4-33.3) for patients without serum viral markers.