Arginine vasopressin (AVP), bombesin, and ACh increase cytosolic free Ca²⁺ and potentiate glucose-induced insulin release by activating receptors linked to phospholipase C (PLC). We examined whether tolbutamide and diazoxide, which close or open ATP-sensitive K⁺ channels (K_ATP channels), respectively, interact with PLC-linked Ca²⁺ signals in HIT-T15 and mouse -cells and with PLC-linked insulin secretion from HIT-T15 cells. In the presence of glucose, the PLC-linked Ca²⁺ signals were enhanced by tolbutamide (3-300 µM) and inhibited by diazoxide (10-100 µM). The effects of tolbutamide and diazoxide on PLC-linked Ca²⁺ signaling were mimicked by BAY K 8644 and nifedipine, an activator and inhibitor of L-type voltage-sensitive Ca²⁺ channels, respectively. Neither tolbutamide nor diazoxide affected PLC-linked mobilization of internal Ca²⁺ or store-operated Ca²⁺ influx through non-L-type Ca²⁺ channels. In the absence of glucose, PLC-linked Ca²⁺ signals were diminished or abolished; this effect could be partly antagonized by tolbutamide. In the presence of glucose, tolbutamide potentiated and diazoxide inhibited AVP- or bombesin-induced insulin secretion from HIT-T15 cells. Nifedipine (10 µM) blocked both the potentiating and inhibitory actions of tolbutamide and diazoxide on AVP-induced insulin release, respectively. In glucose-free medium, AVP-induced insulin release was reduced but was again potentiated by tolbutamide, whereas diazoxide caused no further inhibition. Thus tolbutamide and diazoxide regulate both PLC-linked Ca²⁺ signaling and insulin secretion from pancreatic -cells by modulating K_ATP channels, thereby determining voltage-sensitive Ca²⁺ influx.