Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (l-?-glutamyl-cysteinyl-glycine) and spermidine to form trypanothione [N 1,N 8-bis(glutathionyl)spermidine], a metabolite involved in defense against chemical and oxidant stress and other biosynthetic functions. In Crithidia fasciculata, trypanothione is synthesized from GSH and spermidine via the intermediate glutathionylspermidine in two distinct ATP-dependent reactions catalyzed by glutathionylspermidine synthetase (GspS; EC 22.214.171.124) and trypanothione synthetase (TryS; EC 126.96.36.199), respectively. Here we have cloned a single copy gene (TcTryS) from Trypanosoma cruziencoding a protein with 61% sequence identity withCfTryS but only 31% with CfGspS.Saccharomyces cerevisiae transformed withTcTryS were able to synthesize glutathionylspermidine and trypanothione, suggesting that this enzyme is able to catalyze both biosynthetic steps, unlike CfTryS. When cultures were supplemented with aminopropylcadaverine, yeast transformants contained glutathionylaminopropylcadaverine and homotrypanothione [N 1,N 9-bis(glutathionyl)aminopropylcadaverine], metabolites that have been previously identified in T. cruzi, but not in C. fasciculata. Kinetic studies on recombinant TcTryS purified fromEscherichia coli revealed that the enzyme displays high-substrate inhibition with glutathione (K m andK i of 0.57 and 1.2 mm, respectively, and k cat of 3.4 s-1), but obeys Michaelis-Menten kinetics with spermidine, aminopropylcadaverine, glutathionylspermidine, and MgATP as variable substrate. The recombinant enzyme possesses weak amidase activity and can hydrolyze trypanothione, homotrypanothione, or glutathionylspermidine to glutathione and the corresponding polyamine.