The role of the Ca²⁺-induced Ca²⁺ release channel (ryanodine receptor) in MIN6 pancreatic \-cells was investigated. An endoplasmic reticulum-targeted "cameleon" was used to report lumenal free Ca²⁺. Depolarization of MIN6 cells with KCl led to release of Ca²⁺ from the endoplasmic reticulum. This endoplasmic reticulum Ca²⁺ release was mimicked by treatment with the ryanodine receptor agonists caffeine and 4-chloro-m-cresol, reversed by voltage-gated Ca²⁺ channel antagonists, and blocked by treatment with antagonistic concentrations of ryanodine. The depolarization-induced rise in cytoplasmic Ca²⁺ was also inhibited by ryanodine, which did not alter voltage-gated Ca²⁺ channel activation. Both endoplasmic reticulum and cytoplasmic Ca²⁺ changes induced by depolarization occurred in a dose-dependent manner. Glucose caused a delayed rise in cytoplasmic Ca²⁺ but no detectable change in endoplasmic reticulum Ca²⁺. Carbamyl choline caused endoplasmic reticulum Ca²⁺ release, a response that was not altered by ryanodine. Taken together, these results provide strong evidence that Ca²⁺-induced Ca²⁺ release augments cytoplasmic Ca²⁺ signals in pancreatic -cells.