Release of excitatory amino acids (EAAs) contributes to neuronal death during cerebral ischemia. EAA release occurs by both synaptic and non-synaptic mechanisms. However, studies in vivo yield conflicting estimates of their relative importance. This disparity may reflect differing degrees of substrate deprivation produced by various in vivo models. We used primary rat astrocyte cultures to establish the relationship between substrate deprivation, energy failure, and non-synaptic EAA release. Combined hypoxia and glycolytic blockade produced severe ATP depletion and EAA release (10-fold). In contrast, hypoxia alone caused only a moderate reduction in ATP and did not induce EAA release. These findings suggest that glycolytic metabolism may be an important factor affecting the magnitude of non-synaptic EAA release during ischemia.