1. A study has made of the effect of (Na+‐K+‐Mg2+)‐activated membrane ATP‐ase inhibitors on the acetylcholine release from the terminals of enteric nerves and from cortical slices. 2. The resting output of acetylcholine from slices of rat cortex was not affected by tetrodotoxin or by noradrenaline, indicating the lack of propagated activity during rest. Furthermore, there was an output of acetylcholine in the absence of Ca. 3. The resting acetylcholine output from cortical slices was increased by (a) addition of ouabain or (b) administration of sodium p‐hydroxymercuribenzoate (PHMB), (c) sodium withdrawal and (d) Ca replacement by Ba+. 4. Omission of Ca in the presence of 1 m M ethyleneglycol‐bis‐(β‐aminoethyl‐ether)‐N,N′‐tetra‐acetic acid (EGTA) did not affect the increase of acetylcholine release by the inhibition of (Na+‐K+‐Mg2+)‐activated ATP‐ase induced by ouabain or by PHMB, but reduced that due to Na removal. 5. Ouabain increased acetylcholine release promptly. 6. Mg‐excess (9·3 m M), noradrenaline and adrenaline were capable of reducing the increase of acetylcholine release from cortical slices evoked by ouabain, PHMB or by Ca replacement by Ba, but not by Na removal. 7. A possible role for (Na+‐K+‐Mg2+)‐activated ATP‐ase in the release of acetylcholine is discussed. It is suggested that the effect of Ca and Mg ions on acetylcholine release might be attributed to their ability to inhibit and activate the membrane ATP‐ase, respectively.