The delayed-rectifier K+ channel Kv2.1 exists in highly phosphorylated somatodendritic clusters. Ischemia induces rapid Kv2.1 dephosphorylation and a dispersal of these clusters, accompanied by a hyperpolarizing shift in their voltage-dependent activation kinetics. Transient modulation of Kv2.1 activity and localization following ischemia is dependent on a rise in intracellular Ca2+and the protein phosphatase calcineurin. Here, we show that neuronal free Zn2+also plays a critical role in the ischemic modulation of Kv2.1. We found that sub-lethal ischemia in cultured rat cortical neurons led to characteristic hyperpolarizing shifts in K+ current voltage dependency and pronounced dephosphorylation of Kv2.1. Zn2+chelation, similar to calcineurin inhibition, attenuated ischemic induced changes in K+ channel activation kinetics. Zn2+chelation during ischemia also blocked Kv2.1 declustering. Surprisingly, we found that the Zn2+rise following ischemia occurred in spite of calcineurin inhibition. Therefore, a calcineurin-independent rise in neuronal free Zn2+ is critical in altering Kv2.1 channel activity and localization following ischemia. The identification of Zn2+ in mediating ischemic modulation of Kv2.1 may lead to a better understanding of cellular adaptive responses to injury.