The effect of external free Ca2+ ([Ca2+]ex) on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of state 3 respiration was studied in dog pancreas and adrenal cortex mitochondria. A method for the preparation of mitochondria from pancreas, free of zymogen granules, was developed. The mitochondria showed high respiratory control values and maintained adequate ADP/O ratios in the course of several hours. Half-maximal stimulation (K0.5) of 2-OGDH activity and state 3 respiration by Ca2+ was decreased twofold by spermine, in both pancreas and adrenal cortex mitochondria incubated with Mg2+; Na+ increased the K0.5 value for Ca2+ in both kinds of mitochondria. The matrix Ca2+ content was enhanced by spermine at different [Ca2+]ex. The Ca2+ gradient ([Ca2+]m/[Ca2+]ex) determined in Fluo 3-loaded pancreas mitochondria was found to be below 1, either in the presence of Na+ or in the absence of spermine. The protonmotive force was increased by 1 microM Ca2+, in state 3 conditions, by 6-8 mV; lower Ca2+ concentrations kept the H+ gradient rather constant. An increase in the spermine/Mg2+ ratio at constant external Ca2+ resulted in stimulation of the 2-OGDH activity and state 3 respiration. The data indicate that matrix Ca2+ may regulate the rate of ATP synthesis in pancreas and adrenal cortex mitochondria, through the modulation of 2-OGDH and the maintenance of a high H+ gradient. In addition to changes in external Ca2+, the variation in the spermine/Mg2+ ratio may also exert control of oxidative phosphorylation.