Entamoeba histolytica is a human pathogen that lacks the capacity to synthesize glutathione but can incorporate it, from the growth media or presumably from the human host, to form trypanothione [N(1), N(8)-bis(glutathionyl)-spermidine conjugate]. This novel thiol compound has previously been found in trypanosomatids, as has its precursor glutathionyl-spermidine, which was originally detected in Escherichia coli. Previously we showed the presence of these two thiol compounds in extracts from cultures of Entamoeba histolytica HK9. Here we report that when Entamoeba histolytica HK9 is grown in a culture medium that lacks glutathione (treated with the enzyme gamma-glutamyl transpeptidase), trypanothione is not formed, although the trophozoites can continue dividing for at least 60 h but at 25% lower cell density. The finding of a trypanothione metabolism in Entamoeba histolytica raises many questions: one concerns the possibility of a phylogenetic relationship, in this respect, with trypanosomatids such as Trypanosoma cruzi, T. brucei and Crithidia fasciculata; another concerns its role in cell metabolism; a third concerns it possible use as a target for a rational drug design strategy against this parasite.