Cerebral artery occlusion produces regions of incomplete ischemia (the ischemic penumbra), which, in the absence of reflow, undergo progressive metabolic deterioration culminating in infarction. The factors causing infarction are not yet established, but progression to cell death is preceded by progressive acidosis, decreasing glucose utilization, and ATP depletion. To identify potential mechanisms of glial death in the ischemic penumbra, astrocytes in culture were subjected to conditions that occur during incomplete ischemia: hypoxia, acidosis, and raised extracellular K+. Neither acidosis (to pH 6.2) nor chemical hypoxia (5 mM azide) alone produced significant astrocyte death or marked ATP depletion. By contrast, hypoxia combined with acidosis caused near-complete ATP depletion by 3.5 h and 70% cell death after 7 h. Glycolytic rate increased during hypoxia alone but decreased during hypoxia with acidosis. Since glycolysis is the sole source of ATP production during hypoxia, acidosis inhibition of glycolysis is a likely cause of the far greater ATP depletion resulting from hypoxia with acidosis. Glutamate uptake was reduced during hypoxia and further reduced during hypoxia with acidosis, consistent with the changes in astrocyte ATP. Glutamate uptake, ATP levels, and glycolytic rate each exhibited reductions that were progressive over 3 h of hypoxia with acidosis, and these changes were accompanied by progressive intracellular acidosis. Since ATP depletion leads to acidosis, and acidosis inhibits glycolysis, these findings suggest a regenerative cycle initiated by the combination of hypoxia with acidosis. This cycle could result in progressive metabolic decline and cell death in the ischemic penumbra.