In this study we investigated in rat hippocampal slices (1) how glucose availability affected tissue acidosis during and after anoxia, (2) whether the onset of anoxic depolarization was associated with a specific pH, (3) whether glycolysis was the major source of acidification before and during anoxic depolarization, and (4) whether improved recovery of synaptic function with elevated glucose levels was related to changes in tissue acidosis. Intracellular pH (pHi) and extracellular pH (pHo) were measured simultaneously before, during, and after anoxia in hippocampal slices bathed in 0, 5, 10, and 15 mM glucose. Slices exposed to 0 mM glucose were given 20 mM sodium lactate as a metabolic substrate. We found that the pHi and pHo at which anoxic depolarization occurred depended upon glucose concentration. We also found that elevated glucose availability increased acidification in both the intracellular and extracellular compartments during anoxia and delayed recovery of pH homeostasis after anoxia. Our results suggest that glycolysis is the primary source of acidosis before the onset of anoxic depolarization, but not during anoxic depolarization. Our results also suggest that moderate increases in acidosis resulting from increased glycolysis are potentially beneficial for anoxic survival.