1. The action of spironolactone, a well-known antagonist of mineralocorticoids, on aldosterone regulation was investigated in normal young men to see whether it also acted as an inhibitor of biosynthesis in the adrenal gland. 2. The action of spironolactone was studied under three different conditions: (a) during 3 days of treatment with spironolactone; (b) during 1 day of combined administration with long acting adrenocorticotropic hormone (ACTH); (c) in the course of a continuous infusion of angiotensin II. 3. Spironolactone did not alter the metabolism of aldosterone or cortisol. 4. Spironolactone administration produced: (a) a marked increase in both aldosterone secretion and plasma renin activity, but no change in the plasma aldosterone/plasma renin activity ratio, the cortisol secretion rate or the plasma corticosterone concentration; (b) no blunting in the response of aldosterone to stimulation by ACTH; (c) no decrease in plasma aldosterone concentration when changes of the endogenous renin activity were prevented by an infusion of angiotensin II. 5. These results do not confirm the considerable inhibition of aldosterone excretion found by others after spironolactone administration to normal men. We observed no inhibition of aldosterone biosynthesis by spironolactone. However, a minimal, short-lived inhibition of biosynthesis cannot be excluded, but this possible action of spironolactone plays at best a minor role in the action of this drug.