Ethionamide (ETA), a prodrug that must undergo metabolic activation to exert its cytotoxic effects, is a second line drug against tuberculosis, a disease that infects more than a third of the world's population. It has been proposed, on the basis of genetic experiments, that ETA is activated in Mycobacterium tuberculosis by the protein encoded by the gene Rv3854c (DeBarber, A. E., Mdluli, K., Bosman, M., Bekker, L.-G., and Barry, C. E., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9677-9682; Baulard, A. R., Betts, J. C., Engohang-Ndong, J., Quan, S., McAdam, R. A., Brennan, P. J., Locht, C., and Besra, G. S. (2000) J. Biol. Chem. 275, 28326-28331). We report here the expression, purification, and characterization of the protein encoded by this gene. Our results establish that the enzyme (EtaA) is an FAD-containing enzyme that oxidizes ETA to the corresponding S-oxide. The S-oxide, which has a similar biological activity as ETA, is further oxidized by EtaA to 2-ethyl-4-amidopyridine, presumably via the unstable doubly oxidized sulfinic acid intermediate. This flavoenzyme also oxidizes thiacetazone, thiobenzamide, and isothionicotinamide and thus is probably responsible, as suggested by the observation of crossover resistance, for the oxidative activation of other thioamide antitubercular drugs.