With the expectation that trypanosomal glutathione (GSH) plays a major protective role against the endogenous oxidant stress that results form high intracellular levels of H2O2, we sought to deplete Trypanosoma brucei brucei of their GSH through inhibition of its biosynthesis. Administration of buthionine sulfoximine (BSO), a reversible inhibitor of gamma-glutamylcysteine synthetase, to parasitemic mice resulted in a progressive decrease in trypanosome GSH content, such that parasites isolated after 5 h or BSO treatment contained 50% of normal values. When BSO administration was continued for 18 h (intraperitoneal injection of 4 mmol/kg every 1.5 h), parasitemias temporarily cleared. When inhibitory plasma levels of BSO were maintained for about 27 h, two out of six infected mice were cured and the rest had significantly prolonged survival. These findings demonstrate the potential value of GSH depletion for the treatment of trypanosomiasis.