Polymorphism in N-acetyltransferases NAT1 and NAT2 may contribute to differences in cancer susceptibility of subjects exposed to alkylating compounds. We developed a robust method for simultaneous determination of these NAT polymorphisms: Reverse line blot (RLB) hybridization, based on PCR followed by allele-specific oligo hybridization. On a membrane, allele-specific oligonucleotide probes of the NAT genes (NAT1*4, *3, *10, *11 and NAT2*4, *5, *6, *7, *12) were applied in lines. After separate amplification of the NAT genes, simultaneous hybridization of these products in lines perpendicular to the lines with oligonucleotide probes was performed, followed by nonradioactive detection. This resulted in hybridization patterns, representing the NAT genotype of an individual. RLB hybridizations were conducted on DNA from 240 Dutch Caucasian participants in an ongoing case-control study on colorectal adenoma (including 126 polyp-free control subjects). Results were in complete agreement with those obtained by commonly used methods, i.e., allele-specific PCR and PCR-RFLP. Allele-frequencies in the polyp-free control group were similar to those described in the literature. RLB hybridization is, however, considerably faster and cheaper than the common assays. Moreover, expansion with allelic variants of other genes is relatively easy, which makes RLB hybridization very useful for multiplex analysis of numerous polymorphisms in epidemiological studies.