1. Intracellular micro-electrode recordings of acinar cell membrane potential and resistance were made from the mouse pancreas superfused in vitro. The acinar cells under investigation were stimulated by micro-iontophoretic ACh application from an extracellular AChCl-filled micro-electrode.2. Passing short-lasting ejecting current pulses through the AChCl-electrode caused acinar cell depolarization when the electrode was in an extracellular position not far (< 50 mum) from an acinus impaled by a KCl micro-electrode. After insertion of the AChCl electrode into a neighbouring acinar cell, electrically coupled to the acinar cell already impaled by the KCl-electrode, ejecting ACh current pulses only affected the membrane potential in a direct electrical manner whereas there was no sign of an effect of ACh on the membrane potential.3. Replacing extracellular chloride by sulphate caused a marked increase in the amplitude of the ACh-evoked depolarization. If the membrane potential was recorded with a KCl electrode ACh continued to evoke very large depolarizations even after more than 1 hr exposure to Cl-free solution. If the membrane potential was recorded with a K-citrate electrode the effect of Cl-removal was only transient. Removal of Na(+) during exposure to Cl-free solution reduced the amplitude of the ACh-evoked depolarization somewhat. Readmission of Cl after more than 1 hr of Cl deprivation caused an immediate reversal of the ACh effect into a hyperpolarization.4. Removal of extracellular Ca(2+) caused a marked reduction in the amplitude of small depolarizations evoked by just suprathreshold doses of ACh, whereas there was very little effect on larger depolarizations evoked by maximal or supramaximal ACh ejections. The effect of Ca removal was fully reversible. Addition of Mn after Ca-deprivation was as efficient as Ca in restoring normal electrophysiological responses to small doses of ACh.5. The acinar cell membrane seems only to be responsive to ACh added to the extracellular side and ACh probably causes an increase in membrane Cl permeability in addition to the previously described effects on Na and K permeability. Ca may be important in determining ACh receptor sensitivity.