The second-line antitubercular drugs thiacetazone (TAZ) and ethionamide (ETA) are bioactivated by the mycobacterial enzyme EtaA. We report here that human flavin-containing monooxygenase 2.1 (FMO2.1), which is expressed predominantly in the lung, catalyzes oxygenation of TAZ. The metabolites generated, the sulfenic acid, sulfinic acid, and carbodiimide derivatives, are the same as those produced by EtaA and human FMO1 and FMO3. Two of the metabolites, the sulfenic acid and carbodiimide, are known to be harmful to mammalian cells. FMO2.1 also catalyzes oxygenation of ETA, producing the S-oxide. We have developed a novel spectrophotometric assay for TAZ oxygenation. The assay was used to determine kinetic parameters for TAZ oxygenation catalyzed by human FMO1, FMO2.1, and FMO3 and by EtaA. Although the K(M) values for the four enzyme-catalyzed reactions are similar, k(cat) and, consequently, k(cat)/K(M) (the specificity constant) for FMO2.1-catalyzed TAZ oxygenation are much higher than those of FMO1, FMO3, or EtaA. This indicates that FMO2.1 is more effective in catalyzing TAZ oxygenation than are the other three enzymes and thus is likely to contribute substantially to the metabolism of TAZ, decreasing the availability of the prodrug to mycobacteria and producing toxic metabolites. Because of a genetic polymorphism, Europeans and Asians lack FMO2.1. However, in sub-Saharan Africa, a region in which tuberculosis is a major health problem, a substantial proportion of individuals express FMO2.1. Thus, our results may explain some of the observed interindividual differences in response to TAZ and ETA and have implications for the treatment of tuberculosis in sub-Saharan Africa.