Abstract The addition of oligomycin in the presence of Ca 2+ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca 2+-induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5–20 μM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca 2+-induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca 2+ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase.