The effect of the 12-kDa isoform of FK-506–binding protein (FKBP)12.0 on cardiac excitation–contraction coupling was studied in adult rabbit ventricular myocytes after transfection with a recombinant adenovirus coding for human FKBP12.0 (Ad-FKBP12.0). Western blots confirmed overexpression (by 2.6±0.4 fold, n=5). FKBP12.0 association with rabbit cardiac ryanodine receptor (RyR2) was not detected by immunoprecipitation. However, glutathione S-transferase pull-down experiments indicated FKBP12.0–RyR2 binding to proteins isolated from human and rabbit but not dog myocardium. Voltage-clamp experiments indicated no effects of FKBP12.0 overexpression on L-type Ca²⁺ current (I_Ca,L) or Ca²⁺ efflux rates via the Na⁺/Ca²⁺ exchanger. Ca²⁺ transient amplitude was also not significantly different. However, sarcoplasmic reticulum Ca²⁺ load was 25% higher in myocytes in the Ad-FKBP12.0 group. The reduced ability of I_Ca,L to initiate sarcoplasmic reticulum Ca²⁺ release was observed over a range of values of sarcoplasmic reticulum Ca²⁺ content, indicating that overexpression of FKBP12.0 reduces the sensitivity of RyR2 to Ca²⁺. Ca²⁺ spark morphology was measured in -escin–permeabilized cardiomyocytes. Ca²⁺ spark amplitude and duration were significantly increased, whereas frequency was decreased in cells overexpressing FKBP12.0. These changes were accompanied by an increased sarcoplasmic reticulum Ca²⁺ content. In summary, the effects of FKBP12.0 overexpression on intact and permeabilized cells were similar to those of tetracaine, a drug known to reduce RyR2 Ca²⁺ sensitivity and distinctly different from the effects of overexpression of the FKBP12.6 isomer. In conclusion, FKBP12.0-RyR2 interaction can regulate the gain of excitation–contraction coupling.