The energy state of rat liver mitochondria on day 10 of cold acclimation, when the body temperature decreases significantly concomitantly with an increase in the content of long-chain acyl-CoAs in the liver, has been studied. State 4 and uncoupling respiration rates increase in parallel; however, the integral potentials of the adenine nucleotide system in the liver diminish. In the presence of oligomycin, ADP (20 and 50 microM) decreases the II(+)-permeability and increases the Ca(2+)-capacity of mitochondria in both control and, in a lesser degree, cold-exposed rats. At 90 microM ADP has the same effect on mitochondria of both groups of rats. Carboxyatractylate abolishes the ADP effect on the mitochondria. In EGTA-containing media carboxyatractylate decreases the respiration rate in oligomycin-treated mitochondria. Palmitoyl-CoA increases the II(+)-permeability of the mitochondrial membrane and decreases the Ca(2+)-capacity of mitochondria. ADP abolished the competitive effects of this long-chain acyl-CoA. Possible involvement of the ATP/ADP antiporter in the thermoregulatory response of liver mitochondria in cold-acclimated rats and the role of fatty acids and long-chain acyl-CoAs in this process are discussed.