The flux of a variety of metabolites, nucleotides and coenzymes across the inner membrane of mitochondria is catalysed by a nuclear-coded superfamily of secondary transport proteins called MCs (mitochondrial carriers). The importance of MCs is demonstrated by their wide distribution in all eukaryotes, their role in numerous metabolic pathways and cell functions, and the identification of several diseases caused by alterations of their genes. MCs can easily be recognized in databases thanks to their striking sequence features. Until now, 22 MC subfamilies, which are well conserved throughout evolution, have been functionally characterized, mainly by transport assays upon heterologous gene expression, purification and reconstitution into liposomes. Given the significant sequence conservation, it is thought that all MCs use the same basic transport mechanism, although they exhibit different modes of transport and driving forces and their substrates vary in nature and size. Based on substrate specificity, sequence conservation and carrier homology models, progress has recently been made in understanding the transport mechanism of MCs by new insights concerning the existence of a substrate-binding site in the carrier cavity, of cytosolic and matrix gates and conserved proline and glycine residues in each of the six transmembrane α-helices. These structural properties are believed to play an important role in the conformational changes required for substrate translocation.