1 Longitudinal muscle-myenteric plexus preparations from the guinea-pig ileum were superfused at a constant rate while isotonic contractions were monitored. 2 The preparations were superfused with [3H]-choline while stimulated supramaximally at 0.1 Hz followed by washout in the presence of hemicholinium-3. The evoked release of the label due to a second 0.1 Hz stimulation in the absence of an anticholinesterase was measured. 3 Evoked efflux of the label was initially fast followed by a slower phase. 4 Morphine reduced the size of the pool and the rate of the initial fast efflux and the size of the pool but not the rate of the slow efflux evoked by supramaximal stimulation. 5 Submaximal stimulation reduced only the size of the pools from which the fast and slow efflux originated. 6 Naloxone reversed the depression of contractions and evoked release produced by morphine. 7 Results suggest that 0.1 Hz stimulation releases [3H]-acetylcholine simultaneous from two pools. The fast release may originate from spontaneously firing units whose rate of discharge is depressed by morphine, while the slow release originates from neurones which do not fire spontaneously and whose threshold to field stimulation is increased by morphine.