To investigate the role of the C18 substituents in the agonist/antagonist properties of mineralocorticoids, the activities of certain C18-substituted progesterone (P) derivatives were examined. These compounds were characterized by an unsaturated side-chain in the case of 18-vinylprogesterone (18VP) and 18-ethynylprogesterone (18EP) and by an enone group in the case of 18-oxo-18-vinylprogesterone (18OVP). P and its 18-substituted derivatives bind to the recombinant human MR (hMR) overexpressed in Sf9 cells with the following hierarchy of affinity: P > aldosterone > 18VP > 18EP >> 18OVP. Functional cotransfection assays in CV-1 cells, using mouse mammary tumor virus promoter as a steroid receptor-inducible DNA target sequence, indicated that the mineralocorticoid activity depends on the nature of the C18 substituent. 18VP and 18EP retained the antimineralocorticoid feature of P, with the following order of activity: P = 18VP > 18EP. The antagonist potency of 18VP was higher (IC50, approximately 10(-8) M) than that of spironolactone (IC50, approximately 7 x 10(-8) M), the most widely used aldosterone antagonist. Interestingly, introducing an oxo function at C18 conferred agonist mineralocorticoid properties; 18OVP behaves as a full agonist (ED50, approximately 10(-7) M) with no antagonist activity. In contrast to what was observed when the three 18-substituted P derivatives acted through hMR, they retained the agonist feature of P through the human P receptor, with the following order of potency: P > 18VP = 18OVP > 18EP. The activity of the 18-substituted P derivatives through the human glucocorticoid receptor was only detected at concentrations higher than 10(-6) M; P and 18VP displayed a partial antagonist activity, whereas 18OVP had a full agonist activity (ED50, approximately 2 x 10(-6) M). Thus, the presence of an oxo group at C18(18OVP) does not change the agonist feature of P through human P receptor, but confers to the ligand an agonist activity through hMR, suggesting that the C18 carbonyl group of aldosterone plays a crucial role in its agonist activity.