Human arylamine N-acetyltransferase (NAT) is known to exist as two isoenzymes, NAT1 and NAT2, with different though overlapping substrate specificities. NAT1 and NAT2 are polymorphic at both genetic and phenotypic levels with four distinct alleles described in Caucasians for NAT1. Though clear genotype/phenotype associations exist for NAT2, the same remains unclear for NAT1. Whole blood taken from 32 individuals were NAT1 genotyped and compared to previously obtained NAT1 activities using p-aminobenzoic acid as a substrate. The NAT1 alleles of one individual, who had low NAT1 activity, were sequenced and compared to the wild type allele NAT1*4. A novel, non-conservative, substitution was present in both alleles at nucleotide position 560 and results in the exchange of an arginine for a glutamine at amino acid position 187. A glutamine is found in NAT2 at amino acid position 187 and has been implicated in substrate binding. This report describes a simple and effective genotyping method which detects the four previously reported NAT1 polymorphisms, and the described novel low acetylating polymorphism, by either NAT1 allele specific-PCR amplification or restriction fragment length polymorphism analysis of PCR amplified products. We suggest that NAT1 genotype/phenotype correlations will become more clear as further allelic variants are determined.