The amino acid sequence of ubiquitin from Entamoeba histolytica, as deduced from a cDNA nucleotide sequence, deviated at six positions from the consensus of all other known ubiquitins (ranging from Trypanosoma cruzi to Homo sapiens). The corresponding residues were scattered over the primary sequence, but came close together on the surface of the folded protein structure. We conclude that (i) E. histolytica branched off very early from the main eukaryotic line, and (ii) this organism may yield clues as to the evolutionary development of the ubiquitin system.