The inheritance of rat liver N-acetyltransferase polymorphism was investigated with reciprocal genetic crosses between slow (NSD/N) and rapid (Peth/N) acetylator strains. Rat liver N-acetyltransferase activity was determined using a spectrophotometric assay which measured the amount of arylamine substrate present after incubation with N-acetyltransferase in vitro. Male N-acetyltransferase activities assayed in liver preparations using p-aminobenzoic acid and p-toluidine as substrates indicate bimodality of the parental strains and unimodality of the F-1 generation; limited data suggest trimodality (not significantly different from a 1:2:1 ratio) of the F-2 generation. Reciprocal crosses of WKY/N, another slow acetylator strain, and the Peth/N strain gave results similar to those of the NSD/N x Peth/N cross. Female N-acetyltransferase activities in all strains studied were lower than male N-acetyltransferase activities, but were similarly distributed in the parental and F-1 generations. The male/female N-acetyltransferase activity ratio was substrate- and genotype-dependent. Results show that regulation of the variation of rat liver N-acetyltransferase activity is consistent with autosomal Mendelian inheritance of two major alleles at a single gene locus.