Phosphatidylinositol-4,5-bisphosphate (PIP₂) is a signaling lipid, and stimulus-induced changes in the plasma membrane PIP₂ concentration regulate e.g. vesicle trafficking and cytoskeletal rearrangements. Its precursor phosphatidylinositol-4-monophosphate (PIP) may also exert a signaling function, but it is unclear if stimulus-induced alterations of PIP passively reflect PIP₂ or are differently regulated. In this study we investigated plasma membrane dynamics of PIP and PIP₂ in insulin-secreting MIN6-cells using evanescent wave microscopy and fluorescent PIP- and PIP₂-binding protein translocation reporters. Activation of phospholipase C with 100 mM of the muscarinic agonist carbachol resulted in a lowering of PIP₂ that was paralleled by a sustained elevation of PIP (12.5 ± 0.7% increase of biosensor fluorescence; n=52). The PIP response was sensitive to phosphatidylinositol-4-kinase inhibition, but did not require rise of the cytoplasmic Ca²⁺ concentration. Elevation of the glucose concentration from 3 to 11 mM induced an increase of PIP (22.2 ± 0.1% fluorescence increase; n=56), often followed by oscillations anti-synchronous to changes in PIP₂. The glucose-induced rise in PIP was suppressed when preventing voltage-gated Ca²⁺ influx, and mimicked by Ca²⁺ entry evoked by depolarizations with KCl or tolbutamide. We conclude that glucose- and carbachol-evoked lowering of PIP₂ in insulin-secreting cells is associated with increased levels of PIP. The demonstration that a PIP binding protein is recruited to the membrane in response to cell stimulation reinforces the idea that plasma membrane PIP by itself can exert a second messenger function.