Here we report that colorectal cancer patients show a markedly higher frequency (3-fold) of wild-type NAT2*4 allele homozygotes than the control population. However, a marked difference in NAT2*4/NAT2*4 genotype frequency associated with the patients gender was observed pointing to a male-specific effect of this genotype as a risk factor in colon cancer. The arylamine-N-acetyltransferase (E.C. 2.3.1.5.) NAT2, a phase II detoxification enzyme, has been implicated in procarcinogen activation, namely from food contained arylamines, cigarette smoking, as well as environmental amines of various types. NAT2 is encoded by a polymorphic gene presenting several allelic variants encoding partially inactive enzymes expressed in human liver and colon. Epidemiological studies based on phenotype determination have long indicated the importance of the NAT2 active phenotype as a susceptibility factor in colorectal cancer. In the present study we investigated the NAT2 allelic frequencies and genotype distribution in a group of 114 unrelated colorectal cancer patients, in parallel with 201 healthy Portuguese subjects. We first demonstrate that the frequency of the wild-type NAT2*4 allele in the Portuguese sample population (23.4%) does not significantly differ from the values described for other Europeans. Besides the 3-fold higher frequency of NAT2*4 homozygotes found in colorectal cancer subjects, the NAT2*4/NAT2*5A compound genotype, known to determine a faster acetylator phenotype than other heterozygotic combinations, also increased by the same order of magnitude. These two genotypes represent 32% of the patients population versus 11% of the healthy controls. Taken together, our results strongly indicate that NAT2 genotype, particularly NAT2*4 allele zygosity, constitutes an individual susceptibility trait associated with sporadic colorectal cancer development, probably due to the local dietary habits in Portugal.