We studied the bradykinin-induced changes in phosphoinositide composition of N1E-115 neuroblastoma cells using a combination of biochemistry, microscope imaging, and mathematical modeling. Phosphatidylinositol-4,5-bisphosphate (PIP₂) decreased over the first 30 s, and then recovered over the following 2–3 min. However, the rate and amount of inositol-1,4,5-trisphosphate (InsP₃) production were much greater than the rate or amount of PIP₂ decline. A mathematical model of phosphoinositide turnover based on this data predicted that PIP₂ synthesis is also stimulated by bradykinin, causing an early transient increase in its concentration. This was subsequently confirmed experimentally. Then, we used single-cell microscopy to further examine phosphoinositide turnover by following the translocation of the pleckstrin homology domain of PLCδ1 fused to green fluorescent protein (PH-GFP). The observed time course could be simulated by incorporating binding of PIP₂ and InsP₃ to PH-GFP into the model that had been used to analyze the biochemistry. Furthermore, this analysis could help to resolve a controversy over whether the translocation of PH-GFP from membrane to cytosol is due to a decrease in PIP₂ on the membrane or an increase in InsP₃ in cytosol; by computationally clamping the concentrations of each of these compounds, the model shows how both contribute to the dynamics of probe translocation.