Zinc is a "trace" metal necessary for proper cellular function, but studies, in multiple cell types, have shown that excess free zinc can be toxic (1, 2). It has also been observed that the intra- and extra-cellular concentrations of labile zinc increase dramatically in models of cerebral ischemia (3, 4). Substantial evidence indicates that mitochondrial dysfunction plays a significant role in neuronal death following ischemia (5), and both mitochondrial dysfunction and increased intracellular zinc concentrations have been associated with increased reactive oxygen species (ROS) production and ultimately apoptosis (6, 7). Zinc, specifically, has been shown to inhibit major mitochondrial enzymes of energy production contributing to mitochondrial dysfunction (8). We adapted our expressible fluorescent zinc biosensor (9) to target the mitochondria of PC12 cells, enabling us to ratiometrically image the mitochondrial matrix concentration of labile zinc even at resting (picomolar) levels. This represents the first such development of a sensor with sensitivity for physiological zinc in the mitochondria. We used this biosensor and our previous sensor, in cells which have undergone oxygen/glucose deprivation (OGD), to measure the "free" zinc concentrations following an ischemic-like event. The data suggests that both the intra-cellular and intra-mitochondrial zinc concentrations increase following OGD, albeit at different times, with the mitochondrial increase preceding the cytosolic increase. Our data raises the possibility that an increase in mitochondrial zinc could contribute to cell death in models of ischemia/reperfusion. 1. Canzoniero, L.M., et al. (1999) Journal of Neuroscience 19:RC31, 1-6. 2. Zodl, B., et al. (2003) Journal of Inorganic Biochemistry 97, 324-330. 3. Tonder, N., et al. (1990) Neuroscience Letters 109, 247-252. 4. Frederickson, C. J., et al. (2006) Experimental Neurology 198, 285-293. 5. Fiskum, G. et al. (2008) Mitochondria and Oxidative Stress in Neurodegenerative Disorders: Annals of the New York Academy of Science. 1147, 129-138 6. Weiss, J. H., et al. (2000) Trends in Pharmacological Sciences 21, 395-401. 7. Jiang, D., et al. (2001) Journal of Biological Chemistry 276, 47524 - 47529. 8. Gazaryan, I.G., et al. (2007) Journal of Biological Chemistry 282, 24373-24380. 9. Bozym, R. A., et al. (2006) ACS Chemical Biology 1, 103-111.