Certain membrane lipids are involved in intracellular signalling processes, among them phosphoinositides and diacylglycerol (DAG). They mediate a variety of functions, including the effects of nutrients and neurohormonal stimuli on insulin secretion from pancreatic β-cells. To ensure specificity of the signal, their concentrations are maintained under tight spatial and temporal control. Here, live-cell imaging techniques were employed to investigate spatio-temporal aspects of lipid signalling in the plasma membrane of insulin-secreting β-cells. The concentration of phosphatidylinositol 4-phosphate [PtdIns(4)P] increased after stimulation with glucose or Gq protein-coupled receptor agonists. The glucose effect was Ca2+-dependent, whereas the receptor response was mediated by isoforms of novel protein kinase C (PKC). The increases in PtdIns(4)P were paralleled by lowerings of the phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] concentration. This relationship was not caused by conversion of PtdIns(4,5)P2 to PtdIns(4)P but rather reflected independent regulation of the two lipids. Stimulation of β-cells with glucose or a high K+ concentration induced pronounced, repetitive increases in plasma-membrane DAG concentration, which were locally restricted and lasted only for a few seconds. This pattern was caused by exocytotic release of ATP, which feedback-activates purinergic P2Y1-receptors and stimulates local phospholipase C-mediated DAG generation. Despite their short durations the DAG spikes triggered local activation of PKC. Novel PKCs were recruited to the plasma membrane both after glucose and muscarinic receptor stimulation. While the glucose-induced translocation was synchronized with DAG spiking, muscarinic stimulation induced sustained elevation of the DAG concentration and stable membrane association of the kinase. Also conventional PKCs translocated to the membrane after glucose and receptor stimulation. The glucose-induced response was complex with sustained membrane association mirroring the cytoplasmic Ca2+ concentration, and superimposed brief recurring translocations caused by DAG. Interruption of the purinergic feedback loop underlying DAG spiking suppressed insulin secretion. Since the DAG spikes reflected exocytosis events, a single-cell secretion assay was established, which allowed continuous recording of secretion dynamics from many cells in parallel over extended periods of time. With this approach it was possible to demonstrate that insulin exerts negative feedback on its own release via a phosphatidylinositol 3,4,5-trisphosphate-dependent mechanism.