The mammalian CNS contains an abundance of chelatable zinc that is sequestered in the vesicles of glutamatergic presynaptic terminals and co-released with glutamate. Considerable Zn(2+) is also released during cerebral ischemia and reperfusion (I/R) although the mechanism of this release has not been elucidated. We report here the real time observation of increase of the concentration of extracellular Zn(2+) ([Zn(2+)](o)), accompanied by a rapid increase of intracellular free Zn(2+)concentration, in the areas of dentate gyrus (DG), CA1 and CA3 in acute rat hippocampus slices during ischemia simulated by deprivation of oxygen and glucose (OGD) followed by reperfusion with normal artificial cerebrospinal fluid. A brief period of OGD caused a sustained increase of [Zn(2+)](o). Subsequent reperfusion with oxygenated medium containing glucose resulted in a further increase of [Zn(2+)](o). Longer periods of OGD caused greater increases of [Zn(2+)](o,) and subsequent reperfusion caused still further increases of [Zn(2+)](o,) regardless of OGD duration. The Zn(2+) chelator CaEDTA (10 mM) significantly reduced the increase of [Zn(2+)] induced by OGD and reperfusion. Significant regional differences of [Zn(2+)](o) over the areas of the DG, CA1 and CA3 were not observed during I/R. Neither sodium channel blockade by tetrodotoxin (2 microM), perfusion with nominally calcium-free medium nor anatomical disassociation of the DG, CA1 and CA3 regions from one another by lesioning affected the increase of [Zn(2+)](o). The non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine methyl ester (1 mM), however, blocked the increase of [Zn(2+)](o) during ischemia and reperfusion. The data indicate the important role of NO in causing the release of Zn(2+) during I/R and suggest that NOS inhibitors may be used to reduce Zn(2+)-induced neuronal injury.