To demonstrate an involvement of ATP-sensitive potassium (K_ATP) channel-independent pathways in the first phase of glucose-stimulated insulin secretion (GSIS) from pancreatic β cells, the time course of GSIS from MIN6 cells was analyzed at 30-s sample intervals. GSIS was biphasic with the first phase being observed 120 to 390 s after glucose addition, peaking at 180 s, and with a shoulder at 240 to 330 s. Both 10 μM diazoxide and 3 μM verapamil completely inhibited tolbutamide- or glibenclamide-induced insulin secretion and suppressed the peak of the first phase of GSIS, but did not result in complete suppression. The shoulder following the peak was suppressed by 1 μM dantrolene. The peak, but not shoulder, disappeared under the extracellular Ca²⁺-free condition. A significant amount of insulin secretion remained even in the combined presence of verapamil and dantrolene. The Na⁺ channel blocker tetrodotoxin (30 nM) nearly completely inhibited the first phase release. These results suggest that the first phase of GSIS from MIN6 cells depends on both Ca²⁺-dependent and -independent mechanisms. The former mechanism includes the extracellular Ca²⁺ influx via L-type voltage-dependent calcium channel and intracellular Ca²⁺ release from endoplasmic reticulum via ryanodine receptors, and the latter mechanism involves the pathways associated with Na⁺ channels.