Mespirenone (CAS 87952-98-5), the delta 1,2-15 beta, 16 beta-methylene derivative of spironolactone, proved to be a potent and quite specific inhibitor of adrenocortical mineralocorticoid synthesis in vitro. At 10(-4) mol/l concentrations, the production of aldosterone as well as its possible precursor 18-OH-corticosterone was inhibited more than 40% (p less than 0.01), whereas corticosterone was elevated highly significantly. This points to a clearcut blockade of 18-hydroxylase on the main pathway of aldosterone synthesis by mespirenone. Decrease of 18-OH-progesterone- and increase of 21-deoxyaldosterone secretion suggests two additional points of interference on an alternate pathway of aldosterone biosynthesis, i.e. 18- and 21-hydroxylation, respectively. Thus, mespirenone causes an effective all-around inhibition of mineralocorticoid synthesis in rat adrenals. On the contrary, androstendione levels remained virtually unchanged indicating that mespirenone exerts no inhibitory effects on androgen synthesis. In conclusion, mespirenone, due to inhibition of mineralocorticoid synthesis in addition to antagonistic effects on the receptor level, is a candidate for the development of a new, potent and specific anti-aldosterone drug.