Vesicular zinc was initially considered the sole source of toxic intraneuronal zinc accumulation in response to acute brain injury, but recent evidence suggests that additional sources also exist. Because metallothioneins (MTs) can bind and release zinc, we examined the possibility that the brain-specific form, MT-III, is such a zinc source. After kainate-induced seizures, cytoplasmic zinc accumulation and neuronal death in the hippocampal CA1 region and the thalamus were substantially lower in Mt3-null mice than in wild-type mice. Furthermore, compared with zinc transporter 3 (Znt3)-null mice, Znt3/Mt3 double-null mice exhibited further reductions in neuronal death in CA1 following kainate-induced seizures. Similar reductions in zinc accumulation and neuronal death in hippocampal CA1 and the dentate gyrus in Mt3-null mice were observed in a sodium nitroprusside model of acute brain injury. In contrast to CA1, more neuronal death occurred after kainate-induced seizures in CA3 of Mt3-null mice. These results suggest that intracellular zinc release from MT-III may contribute substantially to zinc-mediated neuronal death in certain brain areas, including the hippocampal CA1 region and the thalamus.