Effects of antimycin A, oligomycin and KCN on tension responses to high K+ or NE and on related Ca++ movements were investigated in rabbit aortic smooth muscle. Selected concentrations of antimycin A(10–6 g/ml), oligomycin (10–6 g/ml) and KCN (1 mM) had little effect on the K+-induced contractile response even though all three agents abolished the increase in La+++-resistant low affinity Ca++ uptake elicited with high K+. Though the mitochondrial inhibitors had varied effects on the NE-induced contractile response, they had no effect on the NE-induced decrease in La+++-resistant high affinity Ca++. Oxygen consumption of the muscle tissue was decreased by antimycin A or oligomycin. These results suggest that the increase in low affinity Ca++ retention elicited with high K+ represents Ca++ uptake by mitochondria. Thus, the correlation between K+-induced uptake of Ca++ and an increased retention of La+++-resistant low affinity Ca++ after exposure to K+ are sequential events that can be dissociated by inhibitors of mitochondrial Ca++ upt