We have used fluo 3-loaded mouse pancreatic acinar cells to investigate the relationship between Ca²⁺ mobilization and intracellular pH (pH_i). The Ca²⁺-mobilizing agonist ACh (500 nM) induced a Ca²⁺ release in the luminal cell pole followed by spreading of the Ca²⁺ signal toward the basolateral side with a mean speed of 16.1 ± 0.3 µm/s. In the presence of an acidic pH_i, achieved by blockade of the Na⁺/H⁺ exchanger or by incubation of the cells in a Na⁺-free buffer, a slower spreading of ACh-evoked Ca²⁺ waves was observed (7.2 ± 0.6 µm/s and 7.5 ± 0.3 µm/s, respectively). The effects of cytosolic acidification on the propagation rate of ACh-evoked Ca²⁺ waves were largely reversible and were not dependent on the presence of extracellular Ca²⁺. A reduction in the spreading speed of Ca²⁺ waves could also be observed by inhibition of the vacuolar H⁺-ATPase with bafilomycin A₁ (11.1 ± 0.6 µm/s), which did not lead to cytosolic acidification. In contrast, inhibition of the endoplasmic reticulum Ca²⁺-ATPase by 2,5-di-tert-butylhydroquinone led to faster spreading of the ACh-evoked Ca²⁺ signals (25.6 ± 1.8 µm/s), which was also reduced by cytosolic acidification or treatment of the cells with bafilomycin A₁. Cytosolic alkalinization had no effect on the spreading speed of the Ca²⁺ signals. The data suggest that the propagation rate of ACh-induced Ca²⁺ waves is decreased by inhibition of Ca²⁺ release from intracellular stores due to cytosolic acidification or to Ca²⁺ pool alkalinization and/or to a decrease in the proton gradient directed from the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ pool to the cytosol.