Mitochondrial permeability transition (MPT) was shown to induce cell death during reperfusion of ischemic myocardium. In the present study, the role of MPT in reperfusion-induced hypercontracture of cardiac myocytes was investigated. To simulate ischemia and reperfusion, isolated cardiac myocytes were superfused anoxically; and then reperfused with a normoxic puffer. The MPT was detected in situ by loading cells with 1 mM calcein. The cytosolic calcein fluorescence was quenched by addition of 1mM CoCL2. Thereafter, calcein fluorescence was only observed in mitochondria. During simulated ischemia no significant change in calcein fluorescence was observed; however, reperfusion induced a marked decline in calcein fluorescence, which indicates an induction of MPT. Concomitantly to MPT induction, cells developed hypercontracture. Application of the MPT inhibitor, cyclosporine (10 mM), prevented the decline in calceine and hypercontracture development (calcein fluorescence as percent of end-anoxic after 3 min reperfusion; control: 70.5±0.2; cyclosporine: 92.7±0.1; n=12; p<0.05); cell length % of end-anoxic after 3 min reperfusion; control: 60.1±2.1; cyclosporine: 97.3±1.9; n=12; p<0.05). Conclusion: Induction of MPT is involved in the reperfusion-induced hypercontracture of cardiac myocytes.