THE EGTA - ruthenium red quench technique was used to obtain initial-velocity plots of Ca2+ uptake by skeletal-muscle mitochondria. The Km was 5 μM and the Hill coefficient 1.9 at both 0° and 10°C. Inorganic phosphate stimulated and Mg2+ inhibited initial rates of transport. In experiments on Ca2+ release, the Na+ Ca2+ exchange was demonstrated. Factors influencing Ca2+ release during anaerobiosis include phosphate concentration and extent of Ca2+ loading. The results are discussed in relation to the possible participation of mitochondria in the calcium-ion regulation of muscle.