Background. In cardiac myocytes, the ryanodine receptor (RyR) is the Ca²⁺-activated channel of the sarcoplasmic reticulum (SR) that releases Ca²⁺ during excitation-contraction coupling. The FK506 binding protein (FKBP12.6) is part of the macromolecular RyR complex. During RyR phosphorylation, dissociation of FKBP12.6 has been reported to enhance RyR open probability. We investigated whether FKBP12.6 overexpression affected SR Ca²⁺ release. Methods. We used a recently developed transgenic mouse (Gellen et al, Circulation, in press) with cardiospecific and conditional overexpression of FKBP12.6 (TG). Intact single cardiomyocytes were enzymatically isolated from the left ventricle of TG and matched wild-type (WT) mice and studied under whole-cell voltage clamp with K5-Fluo-3 as Ca²⁺ indicator. Ca²⁺ transients and sparks were measured during field stimulation after incubation with Fluo-4 AM (5 µM, 20 min). Data were normalized as F/F0. All experiments were done at 35°C. Data are shown as mean ± SEM. Results. The amplitude of the [Ca²⁺]_i transient, measured during field stimulation at 1, 2 and 4 Hz, was comparable between TG and WT (F/F0 at 1 Hz, 2.5±0.2 in TG, 32 cells vs 3.0±0.5 in WT, 35 cells). L-type Ca²⁺ current density was also not different between TG and WT (at +10 mV, -5.8±0.6 pA/pF in TG, 29 cells vs -6.4±0.6 pA/pF in WT, 32 cells). SR Ca²⁺ content, measured by integrating the Na⁺/Ca²⁺ exchange (NCX) current during fast application of 10 mM caffeine, was significantly larger in TG (1.8±0.1 pC/pF, 22 cells vs 1.3±0.1 pC/pF in WT, 37 cells, P<0.05). Fractional Ca²⁺ release, expressed as the ratio between peak [Ca²⁺]_i during a step to +10 mV and peak caffeine-evoked [Ca²⁺]_i, was significantly decreased in TG (47.4±5.0 % in TG, 14 cells vs 62.7±4.9 % in WT, 33 cells, P<0.05). NCX function, measured by the rate of Ca²⁺ decline during caffeine-induced Ca²⁺ release, was significantly decreased (tau 1146.2±76.7 ms in TG, 14 cells vs 774.9±75.4 ms in WT, 33 cells, P<0.05). RyR function was also evaluated by the properties of Ca²⁺ sparks occurring at rest and after loading the SR at 1, 2 and 4 Hz. Spark frequency was not different between WT and TG (after 1 Hz, 0.6±0.04 sparks/100µm/s, 32 cells vs 0.6±0.1 sparks/100µm/s in WT, 34 cells). Data at 2 and 4 Hz were also not different between groups. Spark amplitude (F/F0=1.5±0.01 in TG vs 1.5±0.03 in WT), spark width (2.4±0.05 µm in TG vs 2.3±0.07 µm in WT) and spark duration (31.9±2.2 ms in TG vs 31.7±1.3 ms in WT) were also comparable. Conclusion. Overexpression of FKBP12.6 reduces the fractional release from the SR but the amplitude of the Ca²⁺ transient is maintained by an increased SR Ca²⁺ content.