Sulfamethoxazole toxicity is putatively initiated by the formation of a hydroxylamine metabolite by cytochromes P450. If this reaction could be inhibited, toxicity may decrease. We have studied--in vitro and in vivo--fluconazole, ketoconazole, and cimetidine as potentially suitable clinical inhibitors of sulfamethoxazole hydroxylamine formation. Both fluconazole and ketoconazole in human liver microsomal incubations competitively inhibited sulfamethoxazole N-hydroxylation, with the inhibitory constant (Ki) values of 3.5 and 6 micromol/L, respectively. Cimetidine exhibited a mixed type of inhibition of sulfamethoxazole hydroxylamine formation in human liver microsomes, with IC 50 values (the concentration required to decrease hydroxylamine formation by 50%) of 80 and 800 micromol/L, the lower value being observed when cimetidine was preincubated with microsomes and reduced nicotinamide adenine dinucleotide phosphate. In an in vivo study in six healthy volunteers the inhibition of the cytochrome P450-mediated generation of the toxic metabolite in the presence of fluconazole was shown by a 94% decrease in the area under the plasma concentration-time curve of sulfamethoxazole hydroxylamine. In contrast, the recovery of hydroxylamine in urine decreased by only 60%. Total clearance of sulfamethoxazole was decreased by 26% by fluconazole, most likely because of the inhibition of unidentified P450 elimination pathways. There was close agreement between the predicted (87%) and observed inhibition (94%) of sulfamethoxazole hydroxylamine formation in vivo. Similarly, there was close agreement between in vivo and in vitro Ki values--1.6 and 3.5 micron/L, respectively.