Previous studies suggest baicalein, in addition to its antioxidant effects, protects against hypoxia/reoxygenation injury via its pro-oxidant properties. We hypothesize that a brief period of baicalein treatment prior to ischemia/reperfusion (I/R) may trigger preconditioning protection via a mitochondrial pro-oxidant mechanism. Using an established chick cardiomyocyte model of I/R, cells were preconditioned with baicalein (10 mu M) for 10 min followed by 10-min wash prior to I/R. Intracellular oxidants were measured using 2', 7'-dichlorofluorescin diacetate (DCFH/DA). Cell viability was assessed by propidium iodide and apoptosis determined by DNA fragmentation. Baicalein induced a transient but significant increase of DCF fluorescence within the 10-min preconditioning period, and led to significant reduction of cell death (38.9 +/- 1.8% vs. 58.7 +/- 1.2% in I/R control, n = 6, p < 0.001) and DNA fragmentation after I/R. Cotreatment with N-acetylcysteine (500 mu M), mitochondrial complex III electron transport chain inhibitor myxothiazol (1 mu M), mitochondrial K-ATP channel blocker 5-hydroxydecanoate-Na (5-HD, 500 mu M) or anion channel inhibitor 4', 4'-diisothiocyanato-stilbene-2, 2'-disulfonic acid (DIDS, 200 mu M) resulted in significant abrogation of oxidant increase during induction as well as the protection conferred by baicalein preconditioning. These results suggest that baicalein preconditioning exhibits significant anti-apoptotic protection against cardiomyocyte I/R injury by mitochondrial oxidant signaling, which was in part mediated by mitochondrial K-ATP channel and anion channel opening.