The disks of the vertebrate retinal rod Outer Segment (OS), devoid of mitochondria, are the site of visual transduction, a very energy demanding process. In a previous proteomic study we reported the expression of the respiratory chain complexes I-IV and the oxidative phosphorylation Complex V (F(1)F(0)-ATP synthase) in disks. In the present study, the functional localization of these proteins in disks was investigated by biochemical analyses, oxymetry, membrane potential measurements, and confocal laser scanning microscopy. Disk preparations, isolated by Ficoll flotation, were characterized for purity. An oxygen consumption, stimulated by NADH and Succinate and reverted by rotenone, antimycin A and KCN was measured in disks, either in coupled or uncoupled conditions. Rhodamine-123 fluorescence quenching kinetics showed the existence of a proton potential difference across the disk membranes. Citrate synthase activity was assayed and found enriched in disks with respect to ROS. ATP synthesis by disks (0.7 micromol ATP/min/mg), sensitive to the common mitochondrial ATP synthase inhibitors, would largely account for the rod ATP need in the light. Overall, data indicate that an oxidative phosphorylation occurs in rod OS, which do not contain mitochondria, thank to the presence of ectopically located mitochondrial proteins. These findings may provide important new insight into energy production in outer segments via aerobic metabolism and additional information about protein components in OS disk membranes.