Dyshomeostasis of cytosolic Zn(2+) is a critical mediator of neuronal damage during excitotoxicity. However, the role of this cation in oligodendrocyte pathophysiology is not well understood. The current study examined the contribution of Zn(2+) deregulation to oligodendrocyte injury mediated by AMPA receptors. Oligodendrocytes loaded with the Zn(2+) -selective indicator FluoZin-3 responded to mild stimulation of AMPA receptors with fast cytosolic Zn(2+) rises that resulted from intracellular release, as they were not blocked by the extracellular Zn(2+) chelator Ca-EDTA. Pharmacological experiments suggested that AMPA-induced Zn(2+) mobilization depends on cytosolic Ca(2+) accumulation, arises from mitochondria and protein-bound pools, and is triggered by mechanisms that do not involve the generation of reactive oxygen species. Moreover, intracellular Zn(2+) rises resulting from AMPA receptor activation seem to be promoted by Ca(2+) -dependent cytosolic acidification. Addition of the cell-permeable Zn(2+) chelator TPEN significantly reduced mitochondrial membrane depolarization, reactive oxygen species production, and cell death by sub-maximal activation of AMPA receptors both in vitro and in situ, suggesting that Zn(2+) deregulation is an important mediator of oligodendrocyte excitotoxicity. These data provide evidence that strategies aimed at maintaining Zn(2+) homeostasis may be useful for the treatment of disorders in which excitotoxicity is an important trigger of oligodendroglial death.