1. We have investigated interactions between intracellular pH (pHi) and the intracellular free calcium concentration ([Ca2+]i) in collagenase‐isolated rat lacrimal acinar cells. The fluorescent dyes fura‐2 and 2',7'‐bis(carboxyethyl)‐5‐carboxyfluorescein (BCECF) were used to measure [Ca2+]i and pHi, respectively. 2. Application of the weak base NH4Cl alkalinized the cytosol and caused a dose‐dependent increase in [Ca2+]i. Trimethylamine (TMA) also alkalinized the cytosol and increased [Ca2+]i. The increase in [Ca2+]i evoked by NH4Cl or TMA was much smaller than that evoked by the secretory agonist acetylcholine (ACh). 3. Application of NH4Cl also increased [Ca2+]i in cells bathed in Ca(2+)‐free medium, indicating that NH4Cl released Ca2+ from an intracellular pool. 4. Ammonium chloride had no effect on [Ca2+]i in cells bathed in Ca(2+)‐free medium if agonist‐sensitive intracellular Ca2+ pools had been depleted with either ACh or the microsomal Ca(2+)‐ATPase inhibitor 2,5‐di(tert‐butyl)hydroquinone. Treatment of cells with NH4Cl in Ca(2+)‐free medium reduced the amount of Ca2+ released by ACh. These results suggest that NH4Cl released Ca2+ from the same intracellular pool released by ACh. 5. Calcium release from the agonist‐sensitive pool was also triggered when the cytosol was alkalinized by removing the weak acid acetate. 6. Ammonium chloride caused a modest increase in inositol phosphate production, suggesting that NH4Cl may have released stored Ca2+ via an increase in the intracellular inositol 1,4,5‐trisphosphate concentration. 7. The increase in [Ca2+]i evoked by NH4Cl was not sustained even in the presence of extracellular Ca2+. In contrast, when a low dose of ACh was used to evoke intracellular Ca2+ release of similar magnitude, sustained Ca2+ entry was observed. 8. Alkalinizing the cytosol appeared to partially inhibit Ca2+ entry triggered by thapsigargin or by ACh. 9. We suggest that alkalinizing the cytoplasm in unstimulated lacrimal acinar cells can release Ca2+ from the intracellular agonist‐sensitive Ca2+ pool. However, releasing stored Ca2+ via alkalinization does not appear to trigger significant Ca2+ entry, perhaps because intracellular alkalinization inhibits either the Ca2+ entry pathway or the mechanism which couples the entry pathway to store depletion.