The pH-sensitivity of transepithelial K+ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pHi) was measured microfluorometrically with the pH-sensitive dye 2',7'-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (Isc), which is a measure for transepithelial K+ secretion, was calculated from measurements of the transepithelial voltage (Vt) and the transepithelial resistance (Rt) in a micro-Ussing chamber. All experiments were conducted in virtually HCO3(-)-free solutions. Under control conditions, pHi was 7.01 +/- 0.04 (n = 18), Vt was 9.1 +/- 0.5 mV, Rt 16.7 +/- 0.09 omega cm2, and Isc was 587 +/- 30 microA/cm2 (n = 49). Addition of 20 mM propionate- caused a biphasic effect involving an initial acidification of pHi, increase in Vt and Isc and decrease in Rt and a subsequent alkalinization of pHi, decrease of Vt and increase of Rt. Removal of propionate- caused a transient effect involving an alkalinization of pHi, a decrease of Vt and Isc and an increase in Rt, pHi in the presence of propionate- exceeded pHi under control conditions. Effects of propionate- on Vt, Rt and Isc were significantly larger when propionate- was applied to the basolateral side rather than to the apical side of the epithelium. The pHi-sensitivity of Isc between pH 6.8 and 7.5 was -1089 microA/(cm2.pH-unit) suggesting that K+ secretion ceases at about pHi 7.6. Acidification of the extracellular pH (pHo) caused an increase of Vt and Isc and a decrease of Rt most likely due to acidification of pHi. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pHo sensitivity of Isc between pH 7.4 and 6.4 was -155 microA/(cm2.pH unit). These results demonstrate that transepithelial K+ transport is sensitive to pHi and pHo and that vestibular dark cells contain propionate- uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K+ channel (IsK) in the apical membrane. Whether the effect of pHi on the IsK channel is a direct or indirect effect remains to be determined.