Phospholipase C (PLC) regulates various cellular processes by catalyzing the formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we have investigated the influence of Ca2+ on receptor-triggered PLC activity in individual insulin-secreting beta-cells. Evanescent wave microscopy was used to record PLC activity using green fluorescent protein (GFP)-tagged PIP2/IP3-binding pleckstrin homology domain from PLCdelta1, and the cytoplasmic Ca2+ concentration ([Ca2+]i) was simultaneously measured using the indicator Fura Red. Stimulation of MIN6 beta-cells with the muscarinic-receptor agonist carbachol induced rapid and sustained PLC activation. By contrast, only transient activation was observed after stimulation in the absence of extracellular Ca2+ or in the presence of the non-selective Ca2+ channel inhibitor La3+. The Ca2+-dependent sustained phase of PLC activity did not require voltage-gated Ca2+ influx, as hyperpolarization with diazoxide or direct Ca2+ channel blockade with nifedipine had no effect. Instead, the sustained PLC activity was markedly suppressed by the store-operated channel inhibitors 2-APB and SKF96365. Depletion of intracellular Ca2+ stores with the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors thapsigargin or cyclopiazonic acid abolished Ca2+ mobilization in response to carbachol, and strongly suppressed the PLC activation in Ca2+-deficient medium. Analogous suppressions were observed after loading cells with the Ca2+ chelator BAPTA. Stimulation of primary mouse pancreatic beta-cells with glucagon elicited pronounced [Ca2+]i spikes, reflecting protein kinase A-mediated activation of Ca2+-induced Ca2+ release via IP3 receptors. These [Ca2+]i spikes were found to evoke rapid and transient activation of PLC. Our data indicate that receptor-triggered PLC activity is enhanced by positive feedback from Ca2+ entering the cytoplasm from intracellular stores and via store-operated channels in the plasma membrane. Such amplification of receptor signalling should be important in the regulation of insulin secretion by hormones and neurotransmitters.