Trypanothione [N1,N8-bis(glutathionyl)spermidine] is a unique metabolite found only in trypanosomatids, where it subsumes many of the functions of GSH in other organisms. In Crithidia fasciculata, two distinct ATP-dependent ligases, glutathionylspermidine synthetase (GspS; EC 18.104.22.168) and trypanothione synthetase (TryS; EC 22.214.171.124), are involved in the synthesis of trypanothione from GSH and spermidine. Both enzymes have been cloned previously, but expression in Escherichia coli produced insoluble and inactive protein. Here we report on the successful expression of soluble (His)6-tagged C. fasciculata GspS in E. coli. Following purification using nickel-chelating affinity chromatography, the tag sequence was removed and the enzyme purified to homogeneity by anion-exchange chromatography. The kinetic parameters of the recombinant enzyme have been determined using a coupled enzyme assay and also by HPLC analysis of end-product formation. Under optimal conditions (0.1M K+-Hepes, pH 7.3) GspS has synthetase activity with apparent Km values for GSH, spermidine and MgATP of 242, 59 and 114μM respectively, and a kcat of 15.5s−1. Glutathionylspermidine is formed as end product and the enzyme lacks TryS activity. Like E. coli GspS, the recombinant enzyme also possesses amidase activity (EC 126.96.36.199), hydrolysing glutathionylspermidine to GSH and spermidine with a kcat of 0.38s−1 and a Km of 500μM. GspS can also hydrolyse trypanothione at about 1.5% of the rate with glutathionylspermidine. A single amino acid mutation (Cys-79→Ala) is shown to ablate the amidase activity without affecting the synthetase activity.