In many cells, endocytotic membrane retrieval is accelerated by Ca2+. The effect of Ca2+ on single endocytotic vesicles and fission pore kinetics was examined by measuring capacitance and conductance changes in small membrane patches of insulin-secreting INS-1 cells. In intact cells, elevation of Ca2+ by glucose stimulation induced a 1.8-fold increase in membrane internalisation. This surprisingly resulted from an increased unitary capacitance of endocytotic vesicles whereas the frequency of endocytosis was unaltered. This effect of glucose was prevented by inhibition of L- or R-type Ca2+ channels. Extracellular (pipette) Ca2+ was found to regulate endocytotic vesicle capacitance in a bimodal manner. Vesicle capacitance was increased at intermediate Ca2+ (2.6 mM), but not at high Ca2+ (10 mM). Similar results were obtained upon direct application of 100 nM and 0.5 mM Ca2+ to the intracellular surface of inside-out excised membrane patches, and in these experiments the increase in vesicle capacitance was prevented by the calcineurin inhibitor deltamethrin. Endocytotic fission pore kinetics were accelerated by Ca2+ in both the intact cells and isolated membrane patches; however, the effect in this case was neither bimodal nor deltamethrin sensitive. Membrane retrieval can therefore be upregulated by a Ca2+-dependent increase in endocytotic vesicle size and acceleration of membrane fission in insulin-secreting INS-1 cells.