Acute pancreatitis is characterized by the pathologic activation of zymogens within pancreatic acinar cells. The process requires a rise in cytosolic Ca²⁺ from undefined intracellular stores. We hypothesized that zymogen activation is mediated by ryanodine receptor (RYR)-regulated Ca²⁺ release, because early zymogen activation takes place in a supranuclear compartment that overlaps in distribution with the RYR. Ca²⁺ signals in the basolateral, but not apical, region of acinar cells observed during supraphysiologic agonist stimulation were dependent on RYR Ca²⁺ release. Inhibition of RYR or depletion of RYR-sensitive Ca²⁺ pools each reduced pathologic zymogen activation in isolated acinar cells, but neither treatment affected amylase secretion. Inhibition of RYR also inhibited zymogen activation in vivo. We propose that Ca²⁺ release from the RYR mediates zymogen activation but not enzyme secretion. The findings imply a role for the RYR in acute pancreatitis.