Background and Purpose: It is well established that Pharmacological Preconditioning (PP), achieved with openers of mitochondrial KATP channels like diazoxide, leads to cardioprotection against subsequent ischemia. However, the changes in Ca2+ homeostasis during PP are poorly understood. Here, we investigate the effects of PP on the L-type Ca2+ channel of the adult heart.Experimental approach: Preincubation with diazoxide (100μM) for 90 min was used to induce PP in two preparations: Isolated hearts from rat Wistar and enzymatically dissociated rat ventricular myocytes. Cardiomyocytes were voltage-clamped to measure L-type Ca2+ currents (ICa) with the whole-cell patch-clamp technique and Ca2+ signals and ROS production were measured with the fluorescent probes, Fluo 3-AM and CM-H2DCFDA, respectively. The levels of the α1c subunit, obtained from diazoxide preconditioned hearts, were measured in the membrane fraction of rat ventricles by Western blot. The ROS scavenger NAC was used to examine the role of ROS on the L-type Ca2+ channel after PP in both preparations.Results: Diazoxide induced PP was accompanied by a significant downregulation of the α1c subunit in the membrane fraction and by a reversible reduction in the amplitude of ICa and Ca2+ transients. These effects were complete within 90min and were prevented by NAC. Diazoxide significantly increased ROS production in cardiomyocytes. The reduction of ICa and Ca2+ transients by PP were prevented by the mitochondrial KATP channel blocker 5-HD.Conclusions: Pharmacological preconditioning induced with diazoxide, leads to downregulation of the α1c subunit of the L-type Ca2+ channel. This reduces the influx of Ca2+ through these channels and may contribute to attenuate the overload of Ca2+ during reperfusion.Grants: CONACYT: 60880 (J.S) and 82667 (M.C.G).