Energy transduction in mitochondria requires the transport of many specific metabolites across the inner membrane of this eukaryotic organelle. We have screened the protein sequence database for proteins homologous to the mitochondrial ATP/ADP exchange carrier, and the homologous proteins found were similarly screened to ensure that all currently sequenced members of the mitochondrial carrier family (MCF) had been identified. Thirty-seven proteins were identified, 28 of which were less than 90% identical to any other sequenced member of the MCF, and the latter proteins fell into 10 clusters or subfamilies as follows: (1) ATP/ADP exchangers of mammals, plants, algae, yeast, and fungi (11 members); (2) a bovine oxoglutarate/malate exchanger (one member); (3) mammalian uncoupling carriers (five members); (4) yeast and mammalian phosphate carriers (three members); (5) MRS proteins that suppress mitochondrial splicing defects in Saccharomyces cerevisiae (two members); (6) a putative peroxysomal carrier of Candida boidinii; (7) a putative solute carrier from the protozoan, Oxytricha fallax; (8) a putative solute carrier from S. cerevisiae; (9) a putative solute carrier from Zea mays, and (10) two putative solute carriers from the mammalian thyroid gland. The specificities of proteins in clusters 5 to 10 are not known. A multiple alignment and an evolutionary tree of the 28 selected members of the MCF were constructed, thus defining the conserved residues and the phylogenetic relationships of the proteins. Hydropathy plots of the homologous regions were determined and averaged, and the average hydropathy plots were evaluated for sequence similarity. These analyses revealed that the six transmembrane spanners exhibited varying degrees of sequence conservation and hydrophilicity. These spanners, and immediately adjacent hydrophilic loop regions, were more highly conserved than other regions of these proteins. All members of the MCF appear to consist of a tripartite structure with each of the three repeated segments being about 100 residues in length. Each repeat contains two transmembrane spanners, the first being more hydrophobic with conserved glycyl and prolyl residues, the second, preceded by a highly conserved glycyl residue, being more hydrophilic with largely conserved hydrophilic residues in certain positions. Five of the six spanners are followed by the largely conserved sequence (D/E)-Hy (K/R)[- = any residue; Hy = a hydrophobic residue]. Based on both intracluster and intercluster statistical comparisons, repeats 1, 2, and 3 are homologous, but repeats 1 are more similar to each other than they are to repeats 2 or 3 or repeats 2 or 3 are to each other.(ABSTRACT TRUNCATED AT 400 WORDS)