Rabbit heart mitochondria contained potassium which could not be removed with four washings of isotonic sucrose or sodium chloride at 0–4°C. Aging, increasing concentrations of potassium, and a number of metabolic poisons either partially or completely inhibited the active transport of potassium into heart mitochondria when these particles were incubated for 15 minutes in air at 37.4° in a medium to which alpha-ketoglutarate and AMP or ATP had been added. Compounds uncoupling oxidative phosphorylation—such as arsenite, 2,4-dinitrophenol, l-thyroxine, calcium chloride, dicumarol, pentachlorophenol and methylene blue—inhibited potassium transport but usually only at relatively high concentrations (10–3 m). With the exception of p-chloromercuribenzoate, neither aging nor metabolic inhibitors prevented the extrusion of water by the mitochondria in the presence of alpha-ketoglutarate and AMP or ATP. Although addition of either ATP or substrate alone to the mitochondrial suspension resulted in a significant increase in the potassium gradient, the latter was much greater when both substrate and ATP (or ADP or AMP) were added together. ADP or AMP alone caused a very slight but probably not significant increase in the potassium gradient and creatine phosphate had no effect.