In vitro models of hypoxic/hypoglycemic injury reveal common mechanisms with glutamate excitotoxicity, but glutamate-induced toxicity in the absence of oxygen has never been directly addressed. Therefore, we assessed neuronal survival and intracellular calcium concentrations ([Ca2+]i) in neonatal hippocampal cultures in response to glutamate in the presence and absence of oxygen. Twenty-four hours of hypoxia alone killed 40% of the initial population, attributable to glutamate receptor-stimulated osmotic lysis. A 5 min glutamate exposure in ambient air killed 80% of the initial population by 24 hr later. When cultures were deprived of oxygen during and for 2–24 hr after excitotoxin exposure, glutamate did not cause additional neuronal death beyond that induced by oxygen depletion alone. Toxicities caused by activation of NMDA, AMPA, or kainate receptors were each ameliorated by oxygen depletion. In the absence of oxygen, glutamate evoked normal increases in [Ca2+]i, indicating that glutamate receptors functioned normally. The glutamate-induced increases in [Ca2+]i were not toxic in the absence of oxygen. In a similar manner, oxygen-depletion prevented neuronal killing by the calcium ionophore, ionomycin. Neuronal death produced by hydrogen peroxide or iron sulfate was not ameliorated by oxygen removal. These oxidants maximally produced only a slow increase in [Ca2+]i as the plasma membrane permeability increased nonspecifically. Therefore, oxygen-based reactions were an essential component of calcium-mediated, delayed neuronal death.