Acetylcholine (ACh) and carbamylcholine (Carb) inhibited Ca-dependent action poten- tials and contractions in ventricular muscle from the avian heart. The inhibition by choUnergic drugs was antagonized by atropine (muscarinic) and occurred by two pathways, "indirect" and "direct." Before hatching, ACh had no effect per se, but it inhibited Ca-dependent action potentials that had been augmented by isoproterenol (ISO). This "indirect" pathway for inhibition was detected as early as the 7th incubation day. Acetylcholine had no effect on basal cyclic AMP content, but it reduced cyclic AMP when the nucleotide had been increased by ISO. After hatching, ACh inhibited Ca-dependent action potentials and contractions not only "indirectly" but also "directly." "Direct" inhibition by ACh occurred in preparations treated with 6-hydroxydopamine (or reserpine) and propranolol, so that endogenous cardiac catecholamines probably were not involved. Further, basal levels of cyclic AMP increased after hatching and "direct" inhibition by ACh was associated with a reduction of cyclic AMP. These results are interpreted in a model in which cyclic AMP modulates the permeability of the membrane to Ca2+and in which GTP regulates the β-adrenergic receptor and adenylate cyclase. It is speculated that "indirect" muscarinic inhibition results from interference with GTP-dependent regu- lation of β-adrenergic receptor/adenylate cyclase interaction and "direct" muscarinic inhibition results from interference with GTP-dependent regulation of adenylate cyclase.Circ Res 46: 353-362, 1980