Abstract The endogenous divalent cations, Ca 2+ and Zn 2+, are both highly toxic upon excessive glutamate triggered intracellular accumulation. Given apparent parallels in their neurotoxic mechanisms, the present study aimed to explore interactions between these cations, by examining effects of moderate intracellular Zn 2+ loading on responses to subsequent Ca 2+ influx. Cortical cultures were briefly exposed to high-K + buffer in the presence or absence of Zn 2+ (50–100 μM), to activate and permit a modestly toxic amount of Zn 2+ to enter through VSCC. After 1 h, the cultures were loaded with fluorescent probes, and 2 h after the Zn 2+ exposure, imaged before and after induction of Ca 2+ entry or addition of other drugs. In Zn 2+ preexposed cultures loaded with the Zn 2+ probe, Newport Green, induction of Ca 2+ entry through either VSCC or NMDA channels induced cytoplasmic release of sequestered Zn 2+. The source of this Ca 2+ dependent intracellular Zn 2+ release appears largely to be mitochondria, as indicated by the ability of the mitochondrial protonophore, FCCP, the mitochondrial uncoupler, dinitrophenol with the K + ionophore, valinomycin, or the inducer of mitochondrial permeability transition (mPT), phenylarsine oxide (PAO), to substitute for NMDA in triggering Zn 2+ release. Suggesting functional consequences of mitochondrial Zn 2+ uptake, Zn 2+ preexposures resulted in long-lasting mitochondrial depolarization (assessed with rhodamine 123), and reduced mitochondrial reactive oxygen species generation (assessed with hydroethidine) in response to subsequent NMDA triggered Ca 2+ influx.