Recent pharmacological studies demonstrate a role for zinc (Zn2+) in shaping intracellular calcium (Ca2+) dynamics and vice versa in excitable cells including neurons and cardiomyocytes. Herein, we sought to examine the dynamic of intracellular release of Ca2+and Zn2+upon modifying excitability of primary rat cortical neurons using electric field stimulation (EFS)in vitro. We show that exposure to EFS with an intensity of 7.69 V/cm induces transient membrane hyperpolarization together with transient elevations in the cytosolic levels of Ca2+and Zn2+ions. The EFS-induced hyperpolarization was inhibited by prior treatment of cells with the K+channel opener diazoxide. Chemical hyperpolarization had no apparent effect on either Ca2+or Zn2+. The source of EFS-induced rise in Ca2+and Zn2+seemed to be intracellular, and that the dynamic inferred of an interplay between Ca2+and Zn2+ions, whereby the removal of extracellular Ca2+augmented the release of intracellular Ca2+and Zn2+and caused a stronger and more sustained hyperpolarization. We demonstrate that Zn2+is released from intracellular vesicles located in the soma, with major co-localizations in the lysosomes and endoplasmic reticulum. These studies further support the use of EFS as a tool to interrogate the kinetics of intracellular ions in response to changing membrane potentialin vitro.