The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic β-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+](i)) in cultured rat and mouse B-cells. The tetracaine-specific [Ca2+](i) rise also occurs in the absence of glucose, or in β-cells depolarized by exposure to a Ca2+-deficient medium (< 1 μM) or elevated [K+]0. Furthermore, tetracaine (≥ 300 μM) depolarized the β-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+](i) rise in cultured rat β-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 μM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic β-cell, and thapsigargin (3 μM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect β-cell insulin secretion.