Objectives: Late preconditioning reduces contractile dysfunction during myocardial stunning. Mechanisms involving adaptation of calcium handling during excitation–contraction coupling to late preconditioning remain to be established. Thus, we investigated whether the late preconditioned myocardium is associated with contractile adaptation and changes in the cardiac ryanodine receptor (RyR2) and its regulatory protein FKBP12.6. Methods: Chronically instrumented conscious dogs (coronary occluder, ultrasonic crystals for sonomicrometry) underwent a 10-min coronary artery occlusion followed by reperfusion. They were studied 24 h later in the late preconditioned state (day 1). Results: Maximal velocity of wall thickening at day 1 was increased as compared to corresponding baseline at day 0 (39 ± 4 vs. 30 ± 3 mm/s, p b 0.05) although systolic wall thickening was similar (2.8 ± 0.2 vs. 2.9 ± 0.2 mm), demonstrating a significant change in left ventricular inotropic state. Intracoronary infusion of ryanodine (0.5–6 μg) induced a dose-dependent decrease in wall thickening. In the late preconditioned state, this negative inotropic response was significantly reduced vs. control state, suggesting changes in sarcoplasmic reticulum (SR) Ca 2+-release through RyR2. Immunoquantification of FKBP12.6 revealed a 2.8 fold ventricular increase after late preconditioning as compared to the control state. The amount of RyR2 and its phosphorylated state were similar and binding experiments did not reveal any alterations in B max or K D for RyR2. Calsequestrin, SERCA2a and phospholamban levels were not altered by late preconditioning. Conclusions: The late preconditioned myocardium is characterized by an adaptation of regional function associated with an increased expression of FKBP12.6. This demonstrates an adaptation of the SR Ca 2+-release through RyR2 during late preconditioning.