Abstract: Monitoring Editor: Janet M. Shaw The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (S. cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome bc1-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc1-COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc1-COX supercomplex is perturbed, whereby a decrease in the III2-IV2 assembly state relative to the III2-IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc1-COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc1-COX-TIM23 supercomplex are also discussed.