The contribution of Ca²⁺ release from intracellular stores to the rise in the free cytosolic Ca²⁺ concentration ([Ca²⁺]_c) triggered by Ca²⁺ influx was investigated in mouse pancreatic -cells. Depolarization of -cells by 45 mM K⁺ (in the presence of 15 mM glucose and 0.1 mM diazoxide) evoked two types of [Ca²⁺]_c responses: a monotonic and sustained elevation, or a sustained elevation superimposed by a transient [Ca²⁺]_c peak (TCP) (40-120 s after the onset of depolarization). Simultaneous measurements of [Ca²⁺]_c and voltage-dependent Ca²⁺ current established that the TCP did not result from a larger Ca²⁺ current. Abolition of the TCP by thapsigargin and its absence in SERCA3 knockout mice show that it is caused by Ca²⁺ mobilization from the endoplasmic reticulum. A TCP could not be evoked by the sole depolarization of -cells but required a rise in [Ca²⁺]_c pointing to a Ca²⁺-induced Ca²⁺ release (CICR). This CICR did not involve IP₃ receptors (IP₃R) because it was resistant to heparin. Nor did it involve ryanodine receptors (RyR) because it persisted after blockade of RyR with ryanodine, and was not mimicked by caffeine, a RyR agonist. Moreover, RYR1-2 mRNA were not found and RYR3 mRNA was only slightly expressed in purified -cells. A CICR could also be detected in a limited number of cells in response to glucose. Our data demonstrate, for the first time in living cells, the existence of an atypical CICR that is independent from IP₃R and RyR. This CICR is prominent in response to a supraphysiological stimulation with high K⁺, but plays little role in response to glucose in non-obese mouse pancreatic -cells.