The oxidation of reduced nicotinamide adenine dinucleotide, malate-pyruvate, and succinate by corn mitochondria in buffered 0.2 m KCl was determined as a function of divalent cations. Ni(2+), Mg(2+), Co(2+), Ca(2+), Mn(2+), Sr(2+), and Ba(2+) stimulated reduced nicotinamide adenine dinucleotide oxidation in the absence of inorganic phosphate, with Ca(2+) and Sr(2+) having the greatest effect. Malate-pyruvate and succinate oxidation was stimulated by Ca(2+), Ba(2+), and Sr(2+), but only in the presence of inorganic phosphate. Ca(2+), Sr(2+), and Ba(2+) produced a simulated state 4 to state 3 transition with all three substrates, but only with malate-pyruvate and succinate was there a return to state 4. The order of divalent cation effectiveness suggests that the rate of water substitution from the cation inner coordination hydration sphere may be a rate-limiting step in certain mitochondrial reactions involving electron transport and phosphorylation.