Sub-lethal activation of cell death processes initiate pro-survival signaling cascades. As intracellular Zn2+ liberation mediates neuronal death pathways, we tested whether a sub-lethal increase in free Zn2+ could also trigger neuroprotection. Neuronal free Zn2+ transiently increased following preconditioning, and was both necessary and sufficient for conferring excitotoxic tolerance. Lethal exposure to NMDA led to a delayed increase in Zn2+ that contributed significantly to excitotoxicity in non-preconditioned neurons, but not in tolerant neurons, unless preconditioning-induced free Zn2+ was chelated. Thus, preconditioning may trigger the expression of Zn2+-regulating processes, which, in turn, prevent subsequent Zn2+-mediated toxicity. Indeed, preconditioning increased Zn2+-regulated gene expression in neurons. Examination of the molecular signaling mechanism leading to this early Zn2+ signal revealed a critical role for protein kinase C (PKC) activity, suggesting that PKC may act directly on the intracellular source of Zn2+. We identified a conserved PKC phosphorylation site at serine-32 (S32) of metallothionein (MT) that was important in modulating Zn2+-regulated gene expression and conferring excitotoxic tolerance. Importantly, we observed increased PKC-induced serine phosphorylation in immunopurified MT1, but not in mutant MT1(S32A). These results indicate that neuronal Zn2+ serves as an important, highly regulated signaling component responsible for the initiation of a neuroprotective pathway.