The genome of Saccharomyces cerevisiae encodes 35 members of a family proteins that transport metabolites and substrates across the inner membranes of mitochondria. They include three isoforms of the ADP/ATP translocase and the phosphate and citrate carriers. At the start of our work, the functions of the remaining 30 members of the family were unknown. We are attempting to identify these 30 proteins by overexpression of the proteins in specially selected host strains of Escherichia coli that allow the carriers to accumulate at high levels in the form of inclusion bodies. The purified proteins are then reconstituted into proteoliposomes where their transport properties are studied. Thus far, we have identified the dicarboxylate, succinate-fumarate and ornithine carriers. Bacterial overexpression and functional identification, together with characterization of yeast knockout strains, has brought insight into the physiological significance of these transporters. The yeast dicarboxylate carrier sequence has been used to identify the orthologous protein in Caenorhabditis elegans and, in turn, this latter sequence has been used to establish the sequence of the human ortholog.