Azido derivatives of long-chain fatty acids, 12-(4-azido-2-nitrophenylamino)dodecanoic acid (N3-NpNH-Lau) and 16-(4-azido-2-nitrophenylamino)hexadecanoic acid (N3-NpNH-Pam), were used to study the mechanism of the protonophoric function of long-chain fatty acids in mitochondrial membranes. N3-NpNH-Lau was found to increase resting-state respiration and decrease the membrane potential in a dose-dependent way in a manner similar to that of the natural fatty acid, myristate. Both effects of N3-NpNH-Lau as well as of the myristate were reversed or prevented by the inhibitor of the mitochondrial ADP/ATP carrier (AAC), carboxyatractyloside. This protective effect of carboxyatractyloside was well expressed in rat heart mitochondria and less so in mitochondria within digitonin-permeabilized Ehrlich ascites tumour cells. Photomodification of Ehrlich ascites tumour mitochondria by ultraviolet irradiation in the presence of N3-NpNH-Lau made them more resistant to the uncoupling effect of myristate, and photomodification of rat heart mitochondria resulted in a strong inhibition of AAC which could not be reversed by serum albumin. Photolabelling of rat heart mitochondria with tritiated N3-NpNH-Pam revealed around 10 labelled bands on SDS/polyacrylamide gel electrophoresis. Based on immunodetection with a specific antibody, one of them, corresponding to 30 kDa, was identified as AAC. Specific interaction of AAC with azido fatty acids was confirmed by a high radiolabelling of this band. The role of fatty acids in fine control of the efficiency of oxidative phosphorylation is discussed.