A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain ~9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10 min stimulation with high K⁺. This stimulus depolarized the cell to –10 mV, elevated cytosolic [Ca²⁺] ([Ca²⁺]_i) from a basal concentration of 130 nM to a peak of 1.3 µM and released 0.5 ng insulin/islet, corresponding to 200–300 granules/cell. The Ca²⁺ transient decayed towards the prestimulatory concentration within ~200 s, presumably reflecting Ca²⁺ channel inactivation. Renewed stimulation with high K⁺ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca²⁺. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.