Phylogenetic analyses of ribosomal RNA sequences have played an important role in the study of early evolution of life. However, Loomis and Smith suggested that the ribosomal RNA tree is sometimes misleading--especially when G+C content differs widely among lineages--and that a protein tree from amino acid sequences may be more reliable. In this study, we analyzed amino acid sequence data of elongation factor-1 alpha by a maximum likelihood method to clarify branching orders in the early evolution of eukaryotes. Contrary to Sogin et al.'s tree of small-subunit ribosomal RNA, a protozoan species, Entamoeba histolytica, that lacks mitochondria was shown to have diverged from the line leading to eukaryotes with mitochondria before the latter separated into several kingdoms. This indicates that Entamoeba is a living relic of the earliest phase of eukaryotic evolution before the symbiosis of protomitochondria occurred. Furthermore, this suggests that, among eukaryotic kingdoms with mitochondria, Fungi is the closest relative of Animalia, and that a cellular slime mold, Dictyostelium discoideum, had not diverged from the line leading to Plantae-Fungi-Animalia before these three kingdoms separated.