We present the first molecular phylogenetic examination of the evolutionary position of retortamonads, a group of mitochondrion-lacking flagellates usually found as commensals of the intestinal tracts of vertebrates. Our phylogenies include small subunit ribosomal gene sequences from six retortamonad isolates-four from mammals and two from amphibians. All six sequences were highly similar (95%-99%), with those from mammals being almost identical to each other. All phylogenetic methods utilized unequivocally placed retortamonads with another amitochondriate group, the diplomonads. Surprisingly, all methods weakly supported a position for retortamonads cladistically within diplomonads, as the sister group to Giardia. This position would conflict with a single origin and uniform retention of the doubled-cell organization displayed by most diplomonads, but not by retortamonads. Diplomonad monophyly was not rejected by Shimodaira-Hasegawa, Kishino-Hasegawa, and expected likelihood weights methods but was marginally rejected by parametric bootstrapping. Analyses with additional phylogenetic markers are needed to test this controversial branching order within the retortamonad + diplomonad clade. Nevertheless, the robust phylogenetic association between diplomonads and retortamonads suggests that they share an amitochondriate ancestor. Because strong evidence indicates that diplomonads have secondarily lost their mitochondria (rather than being ancestrally amitochondriate), our results imply that retortamonads are also secondarily amitochondriate. Of the various groups of eukaryotes originally suggested to be primitively amitochondriate under the archezoa hypothesis, all have now been found to have physical or genetic mitochondrial relics (or both) or form a robust clade with an organism with such a relic.