Although there is a general agreement that the protist Entamoeba histolytica lacks glutathione, it has been a matter of dispute as to whether this human parasite contains the glutathione derivative known as trypanothione. In the present study, we describe a gene for the TR (trypanothione reductase) obtained from E. histolytica by PCR amplification of its DNA. After Northern-blot analysis, the radiolabelled DNA probe from Trypanosoma cruzi hybridizes with the total RNA of Entamoeba, showing that the TR gene is expressed as mRNA. We have demonstrated the presence of the NADPH-dependent TR activity in vitro with partially purified extracts and showed also that the thiol-bimane compound isolated and purified from E. histolytica trophozoites, unequivocally corresponds, by matrix-assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) with m/z 1104.4 and the trypanothione-(bimane) with m/z 914.3. The PCR product consisted of 1476 bp (491 deduced amino acids), has sequences diagnostic for the reducing catalytic site (CVNVGC) as well as domains for binding NADPH, FAD I and FAD II that are present in all members of this group of disulphide-reducing enzymes, as well as those unique to TRs. The putative protein sequence is 86% identical with that of TR from T. cruzi and it is also clearly distinguishable from other related reductases by phylogenetic analysis. We can conclude, from these highly reliable experiments, that E. histolytica contains the TR enzyme and the thiol compound trypanothione that was previously supposed to occur only in trypanosomatids.