To examine mitochondrial function in malignant hyperthermia, calcium binding and respiratory functions were measured at 37 C in the absence and presence of halothane in mitochondria isolated from the semitendinous muscle of normal and genetically susceptible swine. Oxygen consumption stimulated by a phosphate acceptor, i.e. adenosine diphosphate (ADP), is state 3 respiration; state 4 respiration is that in the absence of ADP. Values for rate of calcium binding and state 3 respiration in susceptible swine were 40 to 60% of those of normal swine. Halothane altered mitochondrial functions in both normal and susceptible swine to a similar degree: with glutamate-malate substrate, halothane markedly inhibited state 3 respiration, had no significant effect on state 4 respiration, and slightly but significantly inhibited the rate of calcium binding. With succinate substrate, halothane slightly inhibited state 3 respiration, slightly stimulated state 4 respiration, and did not alter calcium binding rate. Oxidation of glutamate-malate provides electrons earlier in the cytochrome chain than does oxidation of succinate. The inhibition by halothane of respiration supported by glutamate-malate that was not seen on respiration supported by succinate suggests a mechanism involving the initial energy reactions in electron transfer. Halothane did not release calcium from actively loaded mitochondria. Dinitrophenol uncoupled state 3 respiratory rates similarly, showing that the lower rate in susceptible swine was not due to a limited capacity of the phosphorylating system. The authors conclude that the reduced respiratory and calcium binding activities in mitochondria from susceptible swine support the diagnosis of a myopathy, but that these do not account for the functional and biochemical derangement observed in clinical malignant hyperthermia.