The decrease of resting MP after denervation in diaphragm muscle of the rat was studied in vitro. Transitory activation of Na+,K+-pump by carbamylcholine, acetylcholine or adrenaline hyperpolarized the muscle fiber membrane but did not compensate the postdenervation fall of the MP. Similarly, the permanent presence of carbamylcholine at culturing media did not prevent the development of changes in the MP after denervation. The decrease of MP was accompanied by spontaneous release of acetylcholine for 24 hrs. Synaptic acetylcholine seems to play no obvious role in neurotrophic control of the resting MP.