Hormonally stimulated secretion of ACTH from AtT-20 mouse pituitary tumor cells is a cyclic AMP-mediated process. The presence of inhibitory cholinergic muscarinic receptors on these cells was recently reported, and in this study, the relationship between the activation of these receptors and the consequent inhibition of cyclic AMP formation and ACTH secretion was investigated. The muscarinic agent, oxotremorine, antagonized both cyclic AMP synthesis and ACTH secretion in response to corticotropin-releasing factor (CRF), vasoactive intestinal peptide, a 27-amino acid peptide with an N-terminal histidine and a C-terminal isoleucine amide, and forskolin. Other muscarinic agents, carbachol and bethanechol, had similar inhibitory effects. The cholinomimetics reduced basal (unstimulated) ACTH secretion without decreasing basal cyclic AMP levels, and also antagonized hormone release in response to cyclic AMP-independent agonists such as K+, A-23187, and phorbol ester. Scopolamine reversed the inhibitory effects of the muscarinic agents on basal and stimulated ACTH secretion and cyclic AMP formation. Increasing the extracellular calcium concentration reversed the muscarinic antagonism of basal and CRF-stimulated hormone release without affecting the cyclic AMP response. Pertussis toxin pretreatment attenuated the inhibitory effects of the muscarinic agents on forskolin-stimulated cyclic AMP synthesis and ACTH secretion as well as the inhibitory effect of carbachol on basal ACTH release. The data suggest that cyclic AMP is an essential mediator in the ACTH secretory pathway, but that an alternate cyclic AMP-independent ACTH pathway also exists in the clonal cells, and that both pathways may be modulated by a common postcholinergic receptor mechanism.