In this paper, we present definitive data to show, from ESI (electrospray ionization) studies, that the thiol-bimane compound isolated and purified from Entamoeba histolytica trophozoites, corresponds unequivocally to the structure of trypanothione. Trypanothione disulphide was shown to have a molecular ion of m/z 722. It was further demonstrated by MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) MS that this thiol compound also corresponds to the characteristic monoprotonated ion of trypanothione-(bimane)(2), which has a molecular ion of m/z 1103.95. The ion pattern of the thiol-bimane compound prepared from the commercial trypanothione standard is identical with the E. histolytica thiol-bimane compound. After HPLC separation, chemical amino acid analysis by dabsylation and dansylation of the thiol bimane compound from Entamoeba showed the presence of the following trypanothione components: glutamic acid, cysteic acid, glycine and spermidine. We can conclude from these highly reliable MS experiments and chemical analyses that E. histolytica contains the thiol compound trypanothione, which was previously thought to occur only in trypanosomatids.