Our aim was to determine if NO prevents mitochondrial oxidant damage by mobilizing intracellular free zinc (Zn 2+ ).Zn 2+ levels were determined by imaging enzymatically isolated adult rat cardiomyocytes loaded with Newport Green DCF. Mitochondrial membrane potential (ΔΨ m ) was assessed by imaging cardiomyocytes loaded with tetramethylrhodamine ethyl ester (TMRE).S-nitroso-N-acetylpenicillamine (SNAP) dramatically increased Zn 2+ , which was blocked by both ODQ and NS2028, two specific inhibitors of guanylyl cyclase. The protein kinase G (PKG) inhibitor KT5823 blocked the effect of SNAP while the PKG activator 8-Br-cGMP mimicked the action of SNAP, indicating that the cGMP/PKG pathway is responsible for the effect of SNAP. The increased Zn 2+ was prevented by 5-hydroxydecanoate (5HD) but was mimicked by diazoxide, implying that mitochondrial K ATP channel opening may account for this effect. Since chelation of Zn 2+ blocked the preventive effect of SNAP on H 2 O 2 -induced loss of ΔΨ m and exogenous zinc (1 μM ZnCl 2 ) prevented dissipation of ΔΨ m , Zn 2+ may play a critical role in the protective effect of NO. The MEK (mitogen-activated protein kinase or extracellular signal-regulated kinase) inhibitor PD98059 blocked the preventive effects of SNAP and zinc on ΔΨ m , indicating that extracellular signal-regulated kinase (ERK) mediates the protective effect of both these compounds on mitochondrial oxidant damage. A Western blot analysis further showed that ZnCl 2 significantly enhances phosphorylation of ERK, confirming the involvement of ERK in the action of Zn 2+ .In isolated cardiomyocytes, NO mobilizes endogenous zinc by opening mitochondrial K ATP channels through the cGMP/PKG pathway. In these cells, Zn 2+ may be an important mediator of the action of NO on the mitochondrial death pathway.