Mitochondrial ADP/ATP carriers catalyse the equimolar exchange of ADP and ATP across the mitochondrial inner membrane, providing metabolic energy for the cell. Crystal structures of the inhibited cytoplasmic-open and matrix-open states support an alternating access mechanism, involving a cytoplasmic and a matrix gate. However, the molecular nature of substrate binding remains unresolved. Substrate binding is concomitant with the structural movements required for translocation, because substrate binding and release provides the free energy changes for the disruption and formation of the gates. The aim of this project was to provide experimental evidence for the residues that are involved in the substrate binding process. For this purpose, all conserved, solvent-accessible residues between the boundaries of the two gates were mutated to alanine, creating a set of 36 variants. A combination of functional complementation, thermostability and transport assays consistently identified five residues (K30, R88, R197, R246 and R287) to be critical for substrate binding. The Ala variants of these residues did not complement growth of an Aac-deficient strain, abolished the concentration-dependent response to substrate, that is observed for the wild-type protein, and had no transport activity. These residues are located roughly in the middle of the central cavity. Around them, another six residues (N85, N96, L135, V138, G192 and Y196) were found to contribute to the binding process. The variants of these residues did not or partially complement growth of the Aac-deficient strain, presented with significantly reduced substrate-induced thermostability shifts compared to the wild-type protein and were either inactive or had altered transport properties. Residues K30, R88, L135, V138, G192, Y196 and R287 cluster together and are accessible with similar conformers in both conformational states, thus forming the main binding site. Residues N96/R197 and N85/R246 form two pairs located on the cytoplasmic and matrix side of the main site and have conformers that change in a state-dependent manner. Hence, they may be involved in the initial binding and guiding of the substrates to the main site and then in their release to the other side of the membrane, inducing conformational changes. The obtained results provide a plausible mechanism for substrate binding, demonstrate that there is a single binding site for ADP and ATP, explain the reversibility of transport and the importance of charge neutralisation in presence of a membrane potential. Results also show that the size and hydrophobicity of the binding pocket are key for the nucleotide base selection.