Comparative studies on the mechanism of action of ACTH1-39 and ACTH5-24 [corticotropin-(1-39)- and corticotropin-(5-24)-peptides] on isolated rat adrenal cells were performed. The relationship between stimulated steroidogenesis and cyclic AMP was very different, suggesting that cyclic AMP does not play the same role in mediating the action of the two agonists. Data from three separate experiments showed that the competitive antagonist ACTH6-24 [corticotropin-(6-24)-peptide] had mean inhibitor constants of 13.4 +/- 3.1 nM against ACTH1-39 and 3.4 +/- 1.0 nM against ACTH5-24, indicating that the steroidogenic actions of these two agonists are mediated by two different receptor types. We conclude that there are two possible mechanisms for corticotropin action, only one of which involves the necessary production of cyclic AMP.