AbstractAlterations in plasma membrane structure and function are considered of primary importance in the pathogenesis of cell injury. Here, fluorescence quenching imaging and Fluorescence Resonance Energy Transfer (FRET) imaging were used to detail alterations of plasma membrane structure during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information and indicated that hypoxic injury leads to a spatially and temporally heterogeneous increase in the rigidity of the plasma membrane. We propose a model, based on our results, for the explanation of alterations in plasma membrane architecture which occur during hypoxic injury. This model also suggests points at which interventional strategies may be applied to prevent or decrease the extent of the hypoxia‐induced changes in membrane structure and thus prolong or even rescue cells from hypoxic injur