Enzyme kinetic parameters of the bioactivation of thiourea-containing compounds by human flavin-containing monooxygenase enzymes (FMOs) FMO1 and FMO3 were investigated. A microtitre-based adaptation of methodology described for the thiourea-dependent oxidation of thiocholine was used to determine the turnover of thiourea-containing compounds by human FMO1 and FMO3. The results show that major differences in enzyme kinetic parameters for N-substituted N'-(4-imidazole-ethyl)thiourea exist between human FMO3 and human FMO1. Whereas Km values of N-substituted N'-(4-imidazole-ethyl)thioureas for human FMO3 are all in the millimolar range, the Km values for human FMO1 range from the low micromolar to the low millimolar range. Furthermore, among a series of N-p-phenyl-substituted N'-(4-imidazole-ethyl)thioureas an interesting structure-activity relationship is evident with both FMO1 and FMO3. Where the Km decreases with increasing electron-withdrawing capacity of the p-substituent in the case of FMO1, the opposite phenomenon may be the case with FMO3. The kcat values of the compounds were all comparable for FMO1, averaging 3.03 +/- 0.56 min-1, whereas more variation was found for FMO3 (3.71 +/- 2.01 min-1). Enzyme kinetic parameters Km and kcat/Km of human FMO1 for N-substituted N'-(4-imidazole-ethyl)thioureas show a high degree of correlation with the results obtained in rat liver microsomes, in which rat FMO1 is the most abundant form, whereas those of human FMO3 do not.