Hybrid cluster protein (HCP) was investigated because of its unique iron-sulfur clusters, which have been found in bacteria and archaea. Here, HCP homologous proteins from the third domain, 'eukarya'(3 amitochondriate protozoans, Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis), were identified. All three amitochondriate protozoan HCPs (GlHCP, EhHCP, and TvHCP) belonged to Class I on the basis of two key characters, the cysteine spacing, Cys-(Xaa)₂Cys(Xaa)_7-8-Cys(Xaa)₅-Cys, and the absence of N-terminal deletion characteristic to the Class III. In phylogenetic analysis performed with amino acid sequences of 3 eukaryal, 5 bacterial, and 4 archaeal HCPs, the maximum likelihood (ML) tree indicated that TvHCP was clustered with Class I HCPs, whereas the other two HCPs (GlHCP and EhHCP) formed an independent clade with a high bootstrapping value (96%) not belonging to any previously recognized HCP class. In spite of the relatively lower bootstrapping value (61%), the position of the new eukaryal GlHCP-EhHCP clade was close to Class I, including the TvHCP, and Classes II and III were closely related with each other. The finding of eukaryal HCPs would help to understand the evolutionary history of HCP.