Biphasic insulin secretion in response to glucose consists of a transient first phase followed by a progressive second phase. It is a well described feature of whole perfused pancreases as well as isolated pancreatic islets of Langerhans. Applying to single cell assays of exocytosis (capacitance monitoring and amperometry) to single canine Beta-cells we have examined the time courses of granule exocytosis in response to voltage-clamp depolarizations that mimic two modes of glucose-induced electrical activity, and then compared these to biphasic insulin secretion. Action potentials evoked in short trains at frequencies similar those recorded during first phase insulin secretion trigger phasic exocytosis from a small pool of insulin granules that are likely docked near voltage-activated Ca²⁺ channels. In contrast, prolonged voltage-clamp pulses mimicking plateau depolarizations occur during second phase insulin secretion and trigger tonic or continuous exocytosis. Comparing the latter results with ones obtained using photorelease of caged Ca²⁺ in other insulin-secreting cells, we suggest that tonic exocytosis likely results from granule release from a highly Ca²⁺-sensitive pool of insulin granules, likely located further from Ca²⁺ channels. Both phasic and tonic modes of exocytosis are enhanced by glucose, via its metabolism. Hence, in canine Beta-cells we propose that two distinct modes of exocytosis, tuned to two types of electrical activity, may underlay biphasic insulin secretion.