Resting state respiration of rat liver mitochondria with succinate or N,N,N',N'-tetrametnyl-p-phenylene-diamine plus ascorbate as substrates was stimulated by myristate. This uncoupling effect was partly reversed not only by carboxyatractyloside and glutamate as reported by others [V. P. Skulachev (1991) FEBS Lett. 294, 158-162; V.N. Samartsev, A.V. Smirnov, I.P. Zeldi, O.V. Markova, E.N. Mokhova, and V.P. Skulachev (1997) Biochim. Biophys. Acta, in press] but also by malonate. Glutamate and malonate also partly restored the mitochondrial membrane potential dissipated by addition of myristate. Myristate inhibited the transport of malonate through the mitochondrial inner membrane, 50% inhibition being reached with 100 nmol fatty acid/mg mitochondrial protein. A similar inhibitory effect was obtained with an azido derivative of long-chain fatty acid, 12-(4-azido-2-nitrophenylamino) dodecanoic acid. This inhibitory effect could be reversed by serum albumin. However, after illumination with ultraviolet light, the inhibition of malonate transport could not be reversed by serum albumin, pointing to photomodification of the dicarboxylate carrier. These results indicate that the dicarboxylate carrier, along with the ADP/ATP carrier and the glutamate carrier, participates in the protonophoric action of fatty acids in mitochondria.