1. Isolated segments of mouse or rat pancreas were placed in a Perspex bath through which physiological saline solutions were circulated. Intracellular recordings from surface acini were made by inserting up to three separate micro-electrodes into neighbouring cells within the same acinus. Through one of these electrodes current pulses could be injected. Electrical coupling between the cells could be continuously assessed. The acinus under investigation was stimulated by iontophoresis of ACh from an extracellular micropipette.2. During resting conditions all cells within an acinus were fully electrically coupled. A 5-10 sec pulse of a relatively large iontophoretic ACh ejecting current, causing initially the well characterized surface cell membrane depolarization and resistance reduction, was followed by a marked electrical uncoupling of neighbouring cells. In some cases one cell was clearly being isolated equally from the two other cells impaled, in other cases the cell into which current pulses were injected was becoming more isolated from one of the neighbouring cells than from the other one.3. The ACh-evoked electrical uncoupling was completely reversible and could be repeatedly observed in the same cell.4. The electrical uncoupling in response to short pulses of ACh (5-10 sec) was not affected by even prolonged (up to 50 min) exposure to Ca-free solution containing EGTA.5. ACh-evoked uncoupling (short pulses) was not reduced about 5 min after introduction of Mn (2 mM), Ni or Co (5 mM). Mn thereafter gradually reduced and finally (after about half an hour) abolished the ACh-evoked uncoupling. Ni and Co gradually enhanced the uncoupling response. The blocking effect of Mn on the ACh-evoked uncoupling occurred much faster in the absence of superfusion fluid Ca.6. During a period of sustained ACh stimulation sustained electrical uncoupling was observed. If Ni or Co (5 mM) was introduced during such a stimulatory period the cells recoupled. This effect of Ni and Co was immediate and fully reversible.7. The uncoupling responses to short pulses of ACh was not reduced immediately after replacing superfusion fluid Na by Tris at a time when the surface cell membrane depolarization and resistance reduction was already severely reduced. Thereafter a gradual slow reduction of the ACh-evoked uncoupling was observed. This did not occur if Na was replaced by Li.8. It is concluded that the Ca needed to raise [Ca(2+)](i) sufficiently to cause electrical uncoupling comes from the cells in cases of uncoupling evoked by short pulses of ACh, but from the external solution in cases of sustained uncoupling evoked by sustained stimulation of ACh.