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Mitochondrial carriers (MCs) are a superfamily of nuclear-encoded proteins that are mostly localized in the inner mitochondrial membrane and transport numerous metabolites, nucleotides, cofactors and inorganic anions. Their unique sequence features, i.e., a tripartite structure, six transmembrane α-helices and a three-fold repeated signature motif, allow MCs to be easily recognized. This review describes how the functions of MCs from Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana (listed in the first table) were discovered after the genome sequence of S. cerevisiae was determined in 1996. In the genomic era, more than 50 previously unknown MCs from these organisms have been identified and characterized biochemically using a method consisting of gene expression, purification of the recombinant proteins, their reconstitution into liposomes and transport assays (EPRA). Information …