Ischemic preconditioning (IP) is a phenomenon in which briefs periods of ischemia activate endogenous mechanisms that protect cardiomyocytes from subsequent ischemia. Pharmacological Preconditioning (PPC) with mitochondrial ATP-sensitive K+ channel (mitoKATP) openers such as diazoxide mimics IP. However, changes in Ca2+ homeostasis during IP and PPC, particularly in Ca2+ channel activity, are poorly understood. We investigated the effects of IP and PPC on cardiac L-type Ca2+ channels.IP was achieved by four cycles of ischemia-reperfusion in isolated hearts. PPC was induced in isolated hearts and in dissociated cardiomyocytes from adult rats by preincubation withdiazoxide. We measured reactive oxygen species (ROS) production and Ca2+ signals associated with action potentials usingfluorescent probes. L-type currents were measured with the whole-cell patch-clamp technique. Levels of the alpha1c subunit of L-type channelsin the cellular membrane were measured by Western blot.IP produced a 20% reduction in alpha1c subunit levels. PPC was accompanied by a 50% reduction in alpha1c subunit levels, and by a reversible fall in L-type current amplitude and Ca2+transients. These effects were prevented by the ROS scavenger N-acetyl-L-cysteine (NAC) or by the mitoKATP channel blocker5-hydroxydecanoate (5-HD). PPC significantly reduced infarct size, an effect blocked by NAC and 5-HD. Nifedipine alsoconferred protection against infarction when applied during the reperfusion period. Downregulation of the alpha1c subunit andCa2+ channel function were prevented in part by the protease inhibitor leupeptin.We conclude that IP andPPC downregulate the alpha1c subunit by a proteolytic process in which ROS are involved. This in turn leads to a reduced Ca2+ influx and attenuates Ca2+ overload during reperfusion.Grants: CONACYT: 60880 (J.S) and 102100 (M.C.G).