It has been demonstrated that K⁺ channel regulation of membrane potential is critical for control of CCK secretion. Because certain K⁺ channels are pH sensitive, it was postulated that pH affects K⁺ channel activity in the CCK-secreting cell line STC-1 and may participate in regulating CCK secretion. The present study examines the role of electroneutral Na⁺/H⁺ exchange on extracellular acidification and hormone secretion. Treatment of STC-1 cells with the amiloride analog ethylisopropyl amiloride (EIPA) to inhibit Na⁺/H⁺ exchange inhibited Na⁺-dependent H⁺ efflux and increased basal CCK secretion. Substituting choline for NaCl in the extracellular medium elevated basal intracellular Ca²⁺ concentration and stimulated CCK release. Stimulatory effects on hormone secretion were blocked by the L-type Ca²⁺ channel blocker diltiazem, indicating that secretion was dependent on the influx of extracellular Ca²⁺. To determine whether the effects of EIPA and Na⁺ depletion were due to membrane depolarization, we tested graded KCl concentrations. The ability of EIPA to increase CCK secretion was inhibited by depolarization induced by 10-50 mM KCl in the bath. Maneuvers to lower intracellular pH (pH_i), including reducing extracellular pH (pH_o) to 7.0 or treatment with sodium butyrate, significantly increased CCK secretion. To examine whether pH directly affects membrane K⁺ permeability, we measured outward currents carried by K⁺, using whole cell patch techniques. K⁺ current was significantly inhibited by lowering pH_o to 7.0. These effects appear to be mediated through changes in pH_i, because intracellular dialysis with acidic solutions nearly eliminated current activity. These results suggest that Na⁺/H⁺ exchange and membrane potential may be functionally linked, where inhibition of Na⁺/H⁺ exchange lowers pH_i and depolarizes the membrane, perhaps through inhibition of pH-sensitive K⁺ channels. In turn, K⁺ channel closure and membrane depolarization open voltage-dependent Ca²⁺ channels, leading to an increase in cytosolic Ca²⁺ and CCK release. The effects of pH_i on K⁺ channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions.