The exocytosis of insulin-containing granules from pancreatic cells is tightly regulated by changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i). We investigated the role of SERCA pump, Na⁺/Ca²⁺ exchanger (NCX) and plasma membrane Ca²⁺-ATPase (PMCA) pump in the Ca²⁺ dynamics of single rat pancreatic cells. When the membrane potential was voltage clamped at -70 mV (in 3 mM glucose at 22 or 35 C), SERCA pump inhibition dramatically slowed (4 fold) cytosolic Ca²⁺ clearance and caused a sustained rise in basal [Ca²⁺]_i via the activation of capacitative Ca²⁺ entry. SERCA pump inhibition increased (1.8 fold) the amplitude of the depolarization-triggered Ca²⁺ transient at 22 C. Inhibition of NCX or PMCA pump had only minor effects on Ca²⁺ dynamics. Simultaneous measurement of [Ca²⁺]_i and exocytosis (with capacitance measurement) revealed that SERCA pump inhibition increased the magnitude of depolarization-triggered exocytosis. This enhancement in exocytosis was not due to the slowing of the cytosolic Ca²⁺ clearance but was closely correlated to the increase in the peak of the depolarization-triggered Ca²⁺ transient. When compared at similar [Ca²⁺]_i with controls, the rise in basal [Ca²⁺]_i during SERCA pump inhibition did not cause any enhancement in the magnitude of the ensuing depolarization-triggered exocytosis. Therefore, we conclude that in rat pancreatic cells, the rapid uptake of Ca²⁺ by SERCA pump limits the peak amplitude of depolarization-triggered [Ca²⁺]_i rise and thus controls the amount of insulin secretion.