Pancreatic duct epithelial cells secretes a HCO₃^–-rich isotonic fluid into the duodenum. Recent evidence suggests that the mechanism of HCO₃^– transport across the apical membrane changes according to the anion composition of the luminal fluid. Even so, it appears to be entirely dependent on the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) (1). When luminal Cl^– concentration is high, intracellular HCO₃^– exits in exchange for luminal Cl^–, probably via one of the SLC26 family anion exchangers, and CFTR works as a Cl^– efflux pathway to maintain the inward Cl^– gradient. However, as the luminal HCO₃^– concentration rises and the luminal Cl^– concentration falls, the apical anion exchangers are no longer able to support HCO₃^– secretion, and CFTR, acting as a HCO₃^– channel, is thought to take over as the principal efflux pathway for HCO₃^– (1). The HCO₃^– concentration of guinea-pig pancreatic juice is 140 mM or more during maximal stimulation, which is similar to human juice and much higher than in juice from mouse and rat pancreas. We previously found that the interlobular duct segments isolated from guinea-pig pancreas secreted HCO₃^– at ~0.5 nmol s^-1 cm^-2 (per unit area of epithelium) following secretin stimulation even when the lumen was filled with a HCO₃^–-rich (125 mM) solution (2). Under these conditions the intracellular concentrations of HCO₃^– and Cl^– were ~20 mM and ~7 mM, respectively, and intracellular potential (V_m) was ~-60 mV (3). There is therefore a luminally-directed electrochemical gradient for HCO₃^– and the low intracellular Cl^– favours HCO₃^– efflux through CFTR. However, to support the observed rate of HCO₃^– secretion, an apical membrane HCO₃^– permeability of 0.25 μm s^-1 would be required (3). In this study we have attempted to determine the apical HCO₃^– conductance by measuring changes in intracellular pH (pH_i) when the cells were de- or hyper-polarized by manipulation of extracellular K⁺ ([K⁺]_B). Interlobular ducts (~100 μm in diameter) were isolated from guinea-pig pancreas by collagenase digestion and microdissection as described previously (2). The lumen was microperfused and pH_i was measured by microfluorometry at 37oC in ducts loaded with the pH-sensitive fluoroprobe BCECF. Dihydro 4,4′ diisothiocyanatostilbene 2,2′ disulphonic acid (H₂DIDS) was used to inhibit basolateral HCO₃^– transport. Changes in [K⁺]_B were achieved by replacement with N-methyl-D-glucamine and extracellular Na⁺ was fixed at 60 mM. Averaged data are presented as the mean ± SEM. Tests for significant differences were made with Student’s t test. (1) Isolated ducts were first superfused with HCO₃^–/CO₂-free Hepes-buffered solution containing H₂DIDS (0.5 mM) and luminally perfused with a solution contaning 125 mM HCO₃^–, 24 mM Cl^–, and 5% CO₂. When [K⁺]_B was raised from 5 to 70 mM, pH_i in unstimulated ducts changed only slightly. During stimulation with dibutyryl AMP (dbcAMP, 0.5 mM), depolarization caused a large increase in pH_i from 6.83 ± 0.11 to 7.32 ± 0.09 (n = 4, p < 0.01). When [K⁺]_B was reduced from 5 to 1 mM, pH_i decreased by 0.11 ± 0.01 (p < 0.05). (2) Experiments were also performed under Cl^–-free conditions with Cl^– replaced by glucuronate. When [K⁺]_B was reduced from 5 to 1 mM and then raised to 70 mM in the presence of dbcAMP, pH_i decreased from 7.15 ± 0.06 (n = 4) to 7.06 ± 0.07 and then increased to 7.54 ± 0.16 (p < 0.05). (3) To calculate the HCO₃^– conductance of the apical membrane, ducts were superfused with the standard HCO₃^–-buffered solution (25 mM HCO₃^–, 5% CO₂) containing H₂DIDS and dbcAMP, and the luminal solution was the same high HCO₃^– solution as previously. Apical membrane HCO₃^– fluxes were calculated from the rate of pH_i change induced by de- or hyperpolarization, taking into account the intracellular buffering capacity (4). The values of V_m, when [K⁺]_B was 1, 5, and 70 mM, were estimated to be -40, -50, and -60 mV using conventional microelectrodes. From the HCO₃^– flux and V_m values (and assuming a cell height of 10 μm), the HCO₃^– permeability coefficient of the apical membrane was calculated to be 0.094 ± 0.016 μm s^-1 (n = 37). De- and hyper-polarization caused changes in pH_i that most probably reflected the influx and efflux of HCO₃^– across the apical membrane. These HCO₃^– movements were not dependent on the presence of Cl^– and were most likely due to a HCO₃^– conductance at the apical membrane, probably CFTR. Our estimate of ~0.1 μm s^-1 for the apical HCO₃^– permeability of guinea-pig duct cells under physiological conditions is close to the value required to account for the secretion of 140 mM HCO₃^– in this species.