Intracellular concentrations of sodium, potassium and calcium together with membrane potentials were measured in cultured murine cortical neurons and glial cells under conditions which mimicked in vivo hypoxia, ischemia and hypoglycemia. These included; glucose omission with and without added pyruvate, addition of rotenone in the presence and absence of glucose and substitution of 2-deoxyglucose for glucose with and without rotenone. Cellular energy levels ([ATP], [ADP], [phosphocreatine], [creatine]) were measured in suspensions of C6 cells incubated in parallel under identical conditions. [Na+]i and [Ca2+]i rose while [K+]i fell and plasma membrane depolarized when energy production was limited. Intracellular acidification was observed when glycolysis was the sole source for ATP synthesis. There was a positive correlation between the extent of energy depletion in glial cells and the magnitude and velocity of alterations in ion levels. Neither glycolysis alone nor oxidative phosphorylation alone were able to ensure unaltered ion gradients. Since oxidative phosphorylation is much more efficient in generating ATP than glycolysis, this finding suggests a specific requirement of the Na pump for ATP generated by glycolysis. Changes in [Na+]i and [K+]i observed during energy depletion were gradual and progressive whereas those in [Ca2+]i were initially slow and moderate with large elevations occurring only as a late event. Increases in [Na+]i were usually smaller than reductions in [K+]i, particularly in the glia, suggestive of cellular swelling. Glia were less sensitive to identical insults than were neurons under all conditions. Results presented in this study lead to the conclusion that the response to energy deprivation of the two main types of brain cells, neurons and astrocytes, is a complex function of their capacity to produce ATP and the activities of various pathways which are involved in ion homeostasis.