1. In freshly prepared and cultured exocrine pancreatic acinar cells from 5- to 7-day-old rats a chloride-selective membrane conductance could be activated by intracellular application of GTPgammaS (40-100 microM), by application of positive pressure (5 cmH2O) to the pipette interior or by challenging the cells with a hyposmolar bath solution. Hyperosmolar bath solutions inhibited the cell volume-sensitive chloride currents. 2. The anion permeability sequence of the cell volume-sensitive chloride conductance was I- > Cl- approximately Br- > F- > methanesulphonate- > glutamate-. I- had a higher permeability but lower conductance than Cl-. The permeability ratio for Pglutamate/PCl was 0.12. 3. The cell volume-sensitive chloride conductance showed outward rectification. Membrane depolarization to high positive voltages (>= +60 mV) caused a time-dependent decay in outward currents. 4. DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) reversibly inhibited the cell volume-sensitive chloride current in a voltage-dependent manner. NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid), quinidine, quinine and tamoxifen caused voltage-independent current inhibition. 5. Combined fura-2 and whole-cell current measurements showed that activation of the cell volume-sensitive chloride current does not involve cytosolic Ca2+ signals. Furthermore, there is no evidence that Ca2+-activated chloride currents play a significant role in cultured pancreatic acinar cells from 5- to 7-day-old rats. 6. Polymerase chain reaction followed by DNA sequence analysis indicated the presence of mRNA homologous to the ClC-3 chloride channel in pancreatic tissue from 5-day-old rats.