Prostaglandins (PGA1, PGE2, PGF2 alpha) were found to increase cholesterol side-chain clevage activity in isolated bovine adrenal cortex mitochondria, provided calcium was present in the incubation medium. Optimal stimulation was observed at low PG concentrations (10-7 to 10-9 M), with malate or malate-NADPH supported side-chain cleavage. Under the same conditions, two endoperoxide analogs and several fatty acids were ineffective. The PG action was not observed with a mitochondrial acetone powder preparation. These observations suggest that primary PG may act by interfering with calcium distribution at the mitochondrial level, leading to the activation of cholesterol side-chain cleavage. Thus, an intracellular action of endogenous PG may be considered in the regulation of adrenal cortex steroidogenic functions.