The effect of some drugs on the release of endogenous acetylcholine from the phrenic nerve-hemidiaphragm preparation of the rat was measured. Muscarinic ligands had no effect. 8-Br-cyclic GMP, a penetrating analogue of cyclic guanosine 3',5'-monophosphate (cyclic GMP) was also without effect. 8-Br-cyclic AMP somewhat enhanced the basal release while the potassium-induced release remained unaltered. In supersensitivity experiments, no specific binding of ligand [3H]-quinuclidinylbenzilate [( 3H]-QNB) was found in homogenates of the diaphragm, either before or after atropine treatment, while concomitant binding studies in the CNS demonstrated the expected increase in muscarinic binding sites after atropine. Our conclusion is that muscarinic acetylcholine receptors are probably absent from the presynaptic motor endplate area of the rat. Certain preliminary results suggest that a presynaptic nicotinic mechanism might be involved in the release of acetylcholine.