The gene encoding the mitochondrial citrate transport protein (CTP) in the yeast Saccharomyces cerevisiae has been identified, and its protein product has been overexpressed in Escherichia coli. The expressed CTP accumulates in inclusion bodies and can be solubilized with sarkosyl. Approximately 25 mg of solubilized CTP at a purity of 75% is obtained per liter of E. coli culture. The function of the solubilized CTP has been reconstituted in a liposomal system where both its kinetic parameters (i.e. Km = 0.36 mM and Vmax = 2.5 mumol/min/mg protein) and its substrate specificity have been determined. Notably, the yeast CTP displays a stricter specificity for tricarboxylates than do CTPs from higher eukaryotic organisms. Dot matrix analysis of the yeast CTP sequence indicates the presence of three homologous sequence domains (each approximately 100 residues in length), which are also related to domains in other CTPs. Thus, the yeast CTP displays the tripartite structure characteristic of other mitochondrial transporters. Alignment of the yeast CTP sequence with CTPs from other sources defines a consensus sequence that displays 89 positions of amino acid identity, as well as the more generalized mitochondrial transporter-associated sequence motif. Based on hydropathy analysis, the yeast CTP contains six putative membrane-spanning alpha-helices. Finally, Southern blot analysis indicates that the yeast genome contains a single gene encoding the mitochondrial CTP. Our data indicate that, based on both its structural and functional properties, the expressed yeast CTP can be assigned membership in the mitochondrial carrier family. The identification of the yeast CTP gene, and the expression and purification of large quantities of its protein product, pave the way for investigations into the roles of specific amino acids in the CTP translocation mechanism, as well as for the initiation of crystallization trials.