Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2in cryo-fixed brain. We demonstrate that PI(4,5)P2forms tiny clusters with a mean size of ∼1000 nm2rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2in neurons.SIGNIFICANCE STATEMENTIn this study, we established an electron microscopic method to visualize and analyze the quantitative distribution pattern of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) on cell membranes using cryo-fixed brain tissues and SDS-digested freeze-fracture replica labeling. PI(4,5)P2interacts with various ion channels and receptors to regulate membrane signaling but its nanoscale distribution and association with these proteins remain elusive. This method revealed PI(4,5)P2clusters preferentially accumulated in specific membrane compartments and its distinct associations with CaV2.1, GIRK3, and mGluR1α in the mouse cerebellum. These results demonstrate usefulness of the method for gaining insights into the physiological functions of PI(4,5)P2.