This study investigated the function of FK506-binding protein (FKBP12.6) using adenoviral-mediated gene transfer to over-express FKBP12.6 (Ad-FKBP12.6) in adult rabbit ventricular cardiomyocytes. Infection with a -galactosidase-expressing adenovirus (Ad-LacZ) was used as a control. Peak-systolic intracellular [Ca²⁺ ] (measured with Fura-2) was higher in the Ad-FKBP12.6 group compared to Ad-LacZ (1Hz field stimulation at 37°C. The amplitude of caffeine-induced Ca²⁺ -release was also greater indicating a higher SR Ca²⁺ content in the Ad-FKBP12.6 group. Voltage clamp experiments indicated that FKBP12.6 over-expression did not change L-type Ca²⁺ current amplitude or Ca²⁺ efflux rates via the Na⁺/Ca²⁺ exchanger. Ca²⁺ transients comparable to those after Ad-FKBP12.6 transfection could be obtained by enhancing SR Ca²⁺ content of Ad-LacZ infected cells with by periods of high frequency stimulation. Line-scan confocal microscopy (Fluo-3 fluorescence) of intact cardiomyocytes stimulated at 0.5Hz (20-21°C) revealed a higher degree of synchronicity of SR Ca²⁺ release and fewer non-responsive CaCa²⁺ release sites in the Ad-FKBP12.6 group compared to control. Ca²⁺ spark morphology was measured in b-escin permeabilised cardiomyocytes at a free [Ca²⁺]_i of 150nmol/L. The average values of the spark parameters (amplitude, duration, width and frequency) were reduced in the Ad-FKBP12.6 group. Increasing intracellular [Ca²⁺] to 400nmol/L caused coherent propagating Ca²⁺ waves in the Ad-FKBP12.6 group but only limited Ca²⁺ release events were recorded in the control group. These data indicate that FKBP12.6 over-expression enhances Ca²⁺ transient amplitude predominately by increasing SR Ca²⁺ content. Moreover, there is evidence that FKBP12.6 can enhance the coupling between SR Ca²⁺ release sites independent of SR content.