Brain mitochondria respiring on glutamate plus malate had a respiratory control index (RCI) for the addition of ADP of 5.5 and P:O ratio of 2.9. These mitochondria also had a Ca2+:O ratio of approximately 5.2, and the ratio of oxygen uptake in the presence of external Ca2+ to that in its absence was 4.0. The reversible transition of respiration to State 3 on the addition of ADP in phosphate media could be blocked by the prior accumulation of 50--200 nmoles/mg of Ca2+. The inhibition was reversible, and on the release of the accumulated Ca2+ by Na+/Ca2+ exchange, the mitochondria became responsive to ADP. The decreased response to ADP following the addition of Ca2+ is probably associated with Ca2+ ions complexing adenine nucleotides within the mitochondria, thus reducing the exchangeable pool and the amount of ADP available to the F1-ATPase.