Trichomonas vaginalis is a unicellular microaerophilic eukaryote that lacks mitochondria yet contains an alternative organelle, the hydrogenosome, involved in pyruvate metabolism. Pathways between the two organelles differ substantially: in hydrogenosomes, pyruvate oxidation is catalysed by pyruvate:ferredoxin oxidoreductase (PFOR), with electrons donated to an [Fe]-hydrogenase which produces hydrogen. ATP is generated exclusively by substrate-level phosphorylation in hydrogenosomes, as opposed to oxidative phosphorylation in mitochondria. PFOR and hydrogenase are found in eubacteria and amitochondriate eukaryotes, but not in typical mitochondria. Analyses of mitochondrial genomes indicate that mitochondria have a single endosymbiotic origin from an alpha-proteobacterial-type progenitor. The absence of a genome in trichomonad hydrogenosomes precludes such comparisons, leaving the endosymbiotic history of this organelle unclear. Although phylogenetic reconstructions of a few proteins indicate that trichomonad hydrogenosomes share a common origin with mitochondria, others do not. Here we describe a novel NADH dehydrogenase module of respiratory complex I that is coupled to the central hydrogenosomal fermentative pathway to form a hydrogenosomal oxidoreductase complex that seems to function independently of quinones. Phylogenetic analyses of hydrogenosomal complex I-like proteins Ndh51 and Ndh24 reveal that neither has a common origin with mitochondrial homologues. These studies argue against a vertical origin of trichomonad hydrogenosomes from the proto-mitochondrial endosymbiont.