Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a phospholipid essential for plasma membrane functions, but its two-dimensional distribution is not clear. Here, we compared the result of sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) of quick-frozen cells with the actual PtdIns(4,5)P2 content and the results obtained by fluorescence biosensor and by labeling of chemically-fixed membranes. In yeast, enrichment of PtdIns(4,5)P2 in the membrane compartment of Can1 (MCC)/eisosome, especially in the curved MCC/eisosome, was evident by SDS-FRL, but not by fluorescence biosensor, GFP-PLC1δ-PH. PtdIns(4,5)P2 remaining after acute ATP depletion and in the stationary phase, 30.0% and 56.6% of the control level, respectively, was not detectable by fluorescence biosensor, whereas the label intensity by SDS-FRL reflected the PtdIns(4,5)P2 amount. In PC12 cells, PtdIns(4,5)P2 was observed in a punctate pattern in the formaldehyde-fixed plasma membrane, whereas it was distributed randomly by SDS-FRL and showed clustering after formaldehyde fixation. The results indicate that the distribution of PtdIns(4,5)P2 can be defined most reliably by SDS-FRL of quick-frozen cells.