Sudden cardiac death resulting from ventricular fibrillation (VF) remains a major cause of mortality. The purpose of this study was to investigate the roles of loss of oxidative phosphorylation and activation of the mitochondrial ATP-sensitive K+ channel and permeability transition pore in VF development during myocardial ischemia by using mitochondrial uncoupling agents (carbonyl cyanide m-chlorophenylhydrazone and 2, 4-dinitrophenol) and channel blockers (5-hydroxydecanoate and cyclosporine A) at concentrations that have been demonstrated to block the intended targets selectively. Isolated rat hearts (n= 8 per group) were perfused with 0.3 μM carbonyl cyanide m-chlorophenylhydrazone, 100 μM 2, 4-dinitrophenol, 0.2 μM cyclosporine A, 100 μM 5-hydroxydecanoate, or vehicle solution and regional ischemia induced after 10 minutes. Carbonyl cyanide m-chlorophenylhydrazone and 2, 4 dinitrophenol caused profound QT shortening and triggered VF in 100% of hearts before ischemia. During ischemia, neither cyclosporine A (88%) nor 5-hydroxydecanoate (100%) reduced VF incidence compared with control (100% VF). In separate hearts, carbonyl cyanide m-chlorophenylhydrazone decreased tissue ATP content, and glibenclamide or glimepiride delayed the QT shortening and onset of VF triggered by carbonyl cyanide m-chlorophenylhydrazone. In conclusion, mitochondrial uncoupling agents trigger VF, likely as a result of ATP depletion with subsequent activation of sarcolemmal ATP-sensitive K+ currents. The mechanism of VF in ischemia does not involve activation of the mitochondrial ATP-sensitive K+ channel or permeability transition pore.