1. The release of acetylcholine (ACh) from Auerbach's plexus of guinea-pig ileum has been measured in eserinized Krebs solution using longitudinal muscle strip preparations. 2. Removal of the external K ions enhanced both the resting and stimulated release of ACh from the plexus. This effect was not affected by tetrodotoxin. 3. On readmission of K+ to tissues which had been suspended in K-free Krebs solution the release of ACh was promptly reduced in both stimulated and unstimulated tissues. The extent of the reduction of ACh release depended on the exposure time to K-free solution, the recovery being delayed by longer exposure. 4. The ACh releasing effect of (1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) was completely inhibited by the readmission of K ions to tissue which had been kept in K-free Krebs solution. 5. Rb+ substitution for K+ produced no change in ACh release and addition of 5-9 mM-Rb after K removal reduced the release of ACh as K did readmission. When the K ions were substituted by Cs+, both the resting and stimulated release were enhanced. The amount of ACh released by a stimulus was enhanced both at low and high frequency of sustained stimulation. 6. Removal of the external K ions increased the release of tritiated noradrenaline (NA), from isolated rat iris; however, when K+ (5-9 mM) was readmitted the release was reduced even below the control value. 7. It is concluded that the stimulation of (Na+-K+)-activated ATP-ase in the membrane inhibits the release of transmitter, and under physiological condition Ca-fluxes and the subsequent inhibition of membrane ATP-ase may be involved in triggering the release of transmitter.