—The FK506-binding protein FKBP12.6 is tightly associated with the cardiac sarcoplasmic reticulum (SR) Ca²⁺-release channel (ryanodine receptor type 2 [RyR2]), but the physiological function of FKBP12.6 is unclear. We used adenovirus (Ad)-mediated gene transfer to overexpress FKBP12.6 in adult rabbit cardiomyocytes. Western immunoblot and reverse transcriptase–polymerase chain reaction analysis revealed specific overexpression of FKBP12.6, with unchanged expression of endogenous FKBP12. FKBP12.6-transfected myocytes displayed a significantly higher (21%) fractional shortening (FS) at 48 hours after transfection compared with Ad-GFP–infected control cells (4.8±0.2% FS versus 4±0.2% FS, respectively; n=79 each; P=0.001). SR-Ca²⁺ uptake rates were monitored in β-escin–permeabilized myocytes using Fura-2. Ad-FKBP12.6–infected cells showed a statistically significant higher rate of Ca²⁺ uptake of 0.8±0.09 nmol/s⁻¹/10⁶ cells (n=8, P<0.05) compared with 0.52±0.1 nmol/s⁻¹/10⁶ cells in sham-infected cells (n=8) at a [Ca²⁺] of 1 μmol/L. In the presence of 5 μmol/L ruthenium red to block Ca²⁺ efflux via RyR2, SR-Ca²⁺ uptake rates were not significantly different between groups. From these measurements, we calculate that SR-Ca²⁺ leak through RyR2 is reduced by 53% in FKBP12.6-overexpressing cells. Caffeine-induced contractures were significantly larger in Ad-FKBP12.6–infected myocytes compared with Ad-GFP–infected control cells, indicating a higher SR-Ca²⁺ load. Taken together, these data suggest that FKBP12.6 stabilizes the closed conformation state of RyR2. This may reduce diastolic SR-Ca²⁺ leak and consequently increase SR-Ca²⁺ release and myocyte shortening.