Sulfonamides are associated with a variety of adverse reactions, some of which have been linked with the classical acetylator phenotypes. Although the slow acetylator phenotype has been identified as a risk factor for hypersensitivity reactions to sulfamethoxazole (SMX), the disposition of this compound appears not to be affected by the acetylation polymorphism in vivo in humans. We therefore investigated the acetylation of SMX by monomorphic (NAT1) and polymorphic (NAT2) arylamine N-acetyltransferases in humans with the objective of determining their role in the metabolism of SMX. SMX was acetylated by both NAT1 and NAT2. Km values determined in hepatic cytosol for NAT1- and NAT2-mediated acetylation of SMX were 1.2 mM and approximately 5 mM, respectively, at an acetyl coenzyme A concentration of 100 microM. Mononuclear leukocytes, which contain only NAT1, had a Km value of 1.2 mM. Km values determined with recombinant NAT1 and NAT2 proteins expressed in Escherichia coli were 1.5 mM and approximately 15 mM, respectively. The higher affinity of NAT1 for SMX indicates that acetylation by this enzyme will predominate at therapeutic plasma concentrations, in agreement with the observed in vivo monomorphic acetylation of SMX. NAT1 may be the primary determinant of SMX systemic metabolic clearance. However, in the hepatocyte NAT2 variation may be an important competitive pathway which influences the extent of oxidative metabolism of SMX to its reactive hydroxylamine metabolite. Therefore, variation in both monomorphic and polymorphic N-acetyltransferases may play a role in determining susceptibility to sulfamethoxazole toxicity.