Entamoeba histolytica was found to grow normally without producing glutathione and the main enzymes of glutathione metabolism, indicating that glutathione is not essential for many eukaryotic processes. This parasitic amoeba is an unusual eukaryote whose special features may help define the crucial functions of glutathione in those eukaryotes that do use it. Since Entamoeba histolytica lacks mitochondria and the usual aerobic respiratory pathways, the finding that it grows without glutathione and other evidence support the hypothesis that a primary function of glutathione in eukaryotes involves protection against oxygen toxicity associated with mitochondria and suggest that eukaryotes may have acquired glutathione metabolism at the time that they acquired mitochondria.