Kinetic parameters for arylamine N-acetyltransferase activity in liver, blood, and bladder from C57BL/6J and A/J mouse strains were determined using an improved assay system, and some deviations were found from previously reported results. In the present studies, blood N-acetyltransferase activity with p-aminobenzoic acid and 2-aminofluorene as substrates was 20- and 10-fold greater, respectively, in C57BL/6J than in A/J mice. Urinary bladder possessed N-acetyltransferase activity for both 2-aminofluorene and p-aminobenzoic acid which differed 2-fold, and reflected the liver and blood phenotype. An apparent Km difference for 2-aminofluorene was observed between C57BL/6J and A/J liver N-acetyltransferase. Contrary to earlier studies, the liver N-acetyltransferase activity differed 3-fold between the A/J and C57BL/6J mouse strains, with either p-aminobenzoic acid or 2-aminofluorene as substrates. Dimethylsulfoxide at concentrations used in the 2-aminofluorene acetylation assay in earlier studies, inhibited the A/J liver N-acetyltransferase to a greater extent than the C57BL/6J enzyme, which may have contributed to the larger difference in liver NAT activity with 2-aminofluorene reported previously.