Whereas previous studies have shown that opening of the mitochondrial K_ATP (mitoK_ATP) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-dayold chick embryos were submitted to anoxia (30 min) followed by reoxygenation (60 min). The chrono-, dromo- and inotropic disturbances, as well as alterations of the electromechanical delay (EMD) reflecting excitation-contraction (E-C) coupling were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe DCFH. The effects of specific mitoK_ATP channel opener diazoxide (DIAZO, 50µM) or blocker 5-hydroxydecanoate (5-HD, 500µM), NOS inhibitor L-NAME (50µM), antioxidant MPG (1mM) and PKC inhibitor chelerythrine (CHEL, 5µM) on oxidative stress and post-anoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of the tested pharmacological agents, alone or in combination. During the first 20 min of postanoxic reoxygenation, DIAZO doubled the peak of ROS production and interestingly accelerated recovery of ventricular EMD and PR interval. The DIAZO-induced ROS production was suppressed by 5-HD, MPG or L-NAME but not by CHEL. The protection of ventricular EMD by DIAZO was abolished by 5-HD, MPG, L-NAME or CHEL whereas the protection of the PR interval was abolished by L-NAME exclusively. Thus, pharmacological opening of the mitoK_ATP channel selectively improves post-anoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS- and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium.