The anaerobic, parasitic protist, Trichomonas vaginalis, is characterized by the absence of mitochondria and the presence of double membrane bound organelles called hydrogenosomes. Succinyl-coenzyme A synthetase is a hydrogenosomal enzyme which catalyzes the formation of ATP via substrate-level phosphorylation. We have characterized genes encoding the alpha subunit of the hydrogenosomal protein succinyl-coenzyme A synthetase (SCS). The alpha-SCS of T. vaginalis is encoded by a multigene family composed of 3 similar genes that do not appear allelic. These 3 alpha-SCS genes encode a protein with a calculated molecular mass of approximately 32.5 kDa that has > 50% identity (> 70% similarity with alpha-SCSs from Escherichia coli, Thermus flavus, and rat liver mitochondria. Antibodies raised against recombinant T vaginalis alpha-SCS expressed in bacteria were used to isolate alpha-SCS proteins from purified hydrogenosomes. These proteins partition into the soluble fraction of hydrogenosomes treated with sodium carbonate at high pH, consistent with a matrix localization in the organelle. Amino-terminal sequencing of purified alpha-SCS proteins shows that mature proteins lack a short, leader sequence of 9 amino acids. These amino terminal sequences which are cleaved from T. vaginalis alpha-SCSs are similar to each other and to all other leader sequences identifed on hydrogenosomal proteins.