We have investigated control mechanisms involved in the propagation of agonist-induced Ca²⁺ waves in isolated mouse pancreatic acinar cells. Using a confocal laser-scanning microscope, we were able to show that maximal stimulation of cells with acetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initial Ca²⁺ release of comparable amounts with both agonists at the luminal cell pole. Subsequent Ca²⁺ spreading to the basolateral membrane was faster with ACh (17.3 ± 5.4 µm/s) than with bombesin (8.0 ± 2.2 µm/s). The speed of bombesin-induced Ca²⁺ waves could be increased up to the speed of ACh-induced Ca²⁺ waves by inhibition of protein kinase C (PKC). Activation of PKC significantly decreased the speed of ACh-induced Ca²⁺ waves but had only little effect on bombesin-evoked Ca²⁺ waves. Within 3 s after stimulation, production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] was higher in the presence of ACh compared with bombesin, whereas bombesin induced higher levels of diacylglycerol (DAG) than ACh. These data suggest that the slower propagation speed of bombesin-induced Ca²⁺ waves is due to higher activation of PKC in the presence of bombesin compared with ACh. The higher increase in bombesin- compared with ACh-induced DAG production is probably due to activation of phospholipase D (PLD). Inhibition of the PLD-dependent DAG production by preincubation with 0.3% butanol led to an acceleration of the bombesin-induced Ca²⁺ wave. In further experiments, we could show that ruthenium red (100 µM), an inhibitor of Ca²⁺-induced Ca²⁺ release in skeletal muscle, also decreased the speed of ACh-induced Ca²⁺ waves. The effect of ruthenium red was not additive to the effect of PKC activation. From the data, we conclude that, following Ins(1,4,5)P₃-induced Ca²⁺ release in the luminal cell pole, secondary Ca²⁺ release from stores, which are located in series between the luminal and the basal plasma membrane, modifies Ca²⁺ spreading toward the basolateral cell side by Ca²⁺-induced Ca²⁺ release. Activation of PKC leads to a reduction in Ca²⁺ release from these stores and therefore could explain the slower propagation of Ca²⁺ waves in the presence of bombesin compared with ACh.