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A single intraperitoneal injection of 2 µg. d-aldosterone monoacetate/g. body weight produced a rapid, but temporary, uncoupling of oxidative phosphorylation in mouse liver mitochondria. This resulted in low P:O ratios in male and female animals of 1·21 and 1·52, respectively. The P:O ratio of females remained somewhat lower than the control levels but there was a progressive improvement in oxidative phosphorylation during the first 24 hr. after the injection leading to P: O ratios similar to those in the controls. Experiments in vitro showed that the uncoupling effects of aldosterone were related to its concentration in the reaction medium. Aldosterone added to fresh rat liver mitochondria, at concentrations of 10–¹⁰, 10–⁷ and 10–⁴m inhibited phosphorylation by 9·5, 77·1 and 95·1% and lowered P:O ratios to 2·46, 1·66 and 0·41, respectively. These changes in oxidative phosphorylation were not related to alteration in ATPase activity and were independent of mitochondrial electrolyte concentration.