Isolated interlobular ducts from the guinea-pig pancreas secrete a HCO3--rich fluid in response to secretin. To determine the role of Cl- transporters in this process, intracellular Cl- concentration ([Cl-]i) was measured in ducts loaded with the Cl--sensitive fluoroprobe, 6-methoxy-N-ethylquinolinium chloride (MEQ). [Cl-]i decreased when the luminal Cl- concentration was reduced. This effect was stimulated by forskolin, was not dependent on HCO3- and was not inhibited by application of the anion channel/transporter inhibitor H2DIDS to the luminal membrane. It is therefore attributed to a cAMP-stimulated Cl- conductance, probably the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. [Cl-]i also decreased when the basolateral Cl- concentration was reduced. This effect was not stimulated by forskolin, was largely dependent on HCO3- and was inhibited by basolateral H2DIDS. It is therefore mediated mainly by Cl-/HCO3- exchange. With high Cl- and low HCO3- concentrations in the lumen, steady-state [Cl-]i was 25-35 mM in unstimulated cells. Stimulation with forskolin caused [Cl-]i to increase by approximately 4 mM due to activation of the luminal anion exchanger. With low Cl- and high HCO3- concentrations in the lumen to simulate physiological conditions, steady-state [Cl-]i was 10-15 mM in unstimulated cells. Upon stimulation with forskolin, [Cl-]i fell to approximately 7 mM due to increased Cl- efflux via the luminal conductance. We conclude that, during stimulation under physiological conditions, [Cl-]i decreases to very low levels in guinea-pig pancreatic duct cells, largely as a result of the limited capacity of the basolateral transporters for Cl- uptake. The resulting lack of competition from intracellular Cl- may therefore favour HCO3- secretion via anion conductances in the luminal membrane, possibly CFTR.