The mitochondrial oxoglutarate carrier exchanges cytosolic malate for 2-oxoglutarate from the mitochondrial matrix. Orthologs of the carrier have a high degree of amino acid sequence conservation, meaning that it is impossible to identify residues important for function on the basis of this criterion alone. Therefore, each amino acid residue in the transmembrane alpha-helices H2 and H6 was replaced by a cysteine in a functional mitochondrial oxoglutarate carrier that was otherwise devoid of cysteine residues. The effects of the cysteine replacement and subsequent modification by sulfhydryl reagents on the initial uptake rate of 2-oxoglutarate were determined. The results were evaluated using a structural model of the oxoglutarate carrier. Residues involved in inter-helical and lipid bilayer interactions tolerate cysteine replacements or their modifications with little effect on transport activity. In contrast, the majority of cysteine substitutions in the aqueous cavity had a severe effect on transport activity. Residues important for function of the carrier cluster in three regions of the transporter. The first consists of residues in the [YWLF]- [KR]-G-X-X-P sequence motif, which is highly conserved in all members of the mitochondrial carrier family. The residues may fulfill a structural role as a helix breaker or a dynamic role as a hinge region for conformational changes during translocation. The second cluster of important residues can be found at the carboxy-terminal end of the even-numbered transmembrane alpha-helices at the cytoplasmic side of the carrier. Residues in H6 at the interface with H1 are the most sensitive to mutation and modification, and may be essential for folding of the carrier during biogenesis. The third cluster is at the midpoint of the membrane and consists of residues that are proposed to be involved in substrate binding.