Spironolactone administration (50 mg/kg/day for 3 days) to make guinea pigs decreased cortisol production by adrenal slices in vitro. Adrenal microsomal and mitochondrial cytochrome P-450 levels were also decreased after treatment with spironolactone. The decline in adrenal cytochrome P-450 content was accompanied by decreases in microsomal 21-hydroxylase and mitochondrial cholesterol side-chain cleavage and 11beta-hydroxylase activities. Activities of other adrenal enzymes, such as delta4-hydrogenase and NADPH-cytochrome c reductase, were unaffected by spironolactone treatment. Cortisone administration to guinea pigs failed to mimic the effects of spironolactone on adrenal function, which indicates specificity of spironolactone action and excludes inhibition of adrenocorticotropin secretion as a mode of action. Addition of spironolactone to isolated adrenal mitochondria or microsomes produced type I spectral changes with spectral dissociation constants similar to those for endogenous steroid substrates. Spironolactone, in vitro, inhibited 11beta- but not 21-hydroxylase activity. The results indicate that spironolactone administration diminishes the activity of adrenal mitochondrial as well as microsomal cytochrome P-450-containing enzymes, resulting in a fall in corticosteroid output.