Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is important for a variety of cellular processes as a precursor for second messengers and by regulating ion channels, the cytoskeleton, and vesicle traffic in many types of cells, including insulin-secreting beta-cells. Here, we applied evanescent wave microscopy and the PIP(2)-binding pleckstrin homology domain from phospholipase C (PLC)-delta fused to the green fluorescent protein to characterize the regulation of plasma membrane PIP(2) in individual insulin-secreting MIN6 beta-cells. Elevation of the glucose concentration from 3 to 11 mmol/l evoked antisynchronous oscillations of [PIP(2)] and cytoplasmic Ca(2+)concentration, consistent with PLC being periodically activated by the voltage-dependent Ca(2+) influx. The effect of adenine nucleotides on [PIP(2)] was studied in cells permeabilized with alpha-toxin. ATP dose- dependently stimulated PIP(2) synthesis with half-maximal effect at 300 mumol/l. Omission of the nucleotide resulted in rapid loss of PIP(2) with t(1/2) < 40 s. ADP also stimulated PIP(2) formation, but this effect reflected local ATP formation and was prevented by the adenylate kinase inhibitor diadenosine-pentaphosphate. The ATP-induced PIP(2) synthesis was counteracted by the ADP analog adenosine-5'-O-2-thiodiphosphate. We conclude that plasma membrane PIP(2) is dynamically regulated by intracellular Ca(2+) and the ATP-to-ADP ratio in insulin-secreting cells. The rapid turnover allows maintenance of PIP(2) levels while generating second messengers of critical importance for insulin secretion.