The mechanism of glucose-induced biphasic insulin release by the B cell was investigated using isolated rat pancreatic islets. In perifusion experiments, 16.7 mM glucose in combination with 25 mM K+ transformed the high K(+)-induced monophasic insulin release into a biphasic one in the presence of diazoxide (Dz), an ATP-sensitive K+ channel opener. Inclusion of Dz during the initial 6 min of glucose stimulation abolished the first phase, but was without effect on the second phase. In batch incubation experiments, fuels, including 16.7 mM glucose, 6 mM D-glyceraldehyde, and 10 mM 2-ketoisocaproate, but not sulfonylurea, caused time-dependent potentiation of the B cell so that the response to 25 mM K+, applied later, was increased in the fuel-primed islets. Inclusion of Dz or lowering extracellular Ca2+ (to micromolar range) during the priming, which eliminates the initiation of insulin release, did not eradicate the potentiation. We conclude that high glucose closes ATP-sensitive K+ channels, leading to membrane depolarization, Ca2+ influx, and initiation of insulin release (first phase), and subsequently self-augments insulin release in an ATP-sensitive K+ channel-independent manner (second phase), acting at steps distal to cytosolic Ca2+ elevation. The biphasic insulin release is thus generated by an interaction of ATP-sensitive K+ channel-dependent and -independent glucose actions.