More than one third of the heart muscle cell volume consists of mitochondria (mito). The constant contractile activity and corresponding high metabolic rate of heart muscle make the heart highly dependent on mito aerob metabolism. Experimental studies of myocardial ischemia strongly indicate that mito play an important role in irreversible ischemic injury and myocardial infarction due to oxygen deprivation and ischemia. Studies were conducted in integrated animal models using pharmacological agents, which do not directly prove mito as target for protection. Direct studies of superfused tetramethylrhodamine loaded isolated adult rat myocytes using confocal microscopy permit studies of mito membrane potential. Resistance towards membrane permeability transition has been tested by subcellular site-specific laser induced ROS exposure of selected rows of mito (Zorov et al J Exp Med 2000, Juhaszova et al 2004). Use of this technique confirm mito cardiomyocyte protection by melatonin, diazoxide and estradiol, agents that reduce irreversible ischemic injury in the intact heart. The results demonstrate protection at the level of single in situ mito by significant delay of permeability transition pore opening and loss of mito membrane potential. Pharmacological preconditioning (pre-treatment followed by washout) indicate that cell survival signalling converge on mito. In conclusion, studies of in situ mito in intact isolated heart myocytes support a strong mechanistic role for protection against permeability transition in a variety of cardioprotection protocols.