Ryanodine receptors (RyRs) are intracellular channels that regulate the release of Ca2+ from the endoplasmic reticulum of many cell types. The RyRs are physically associated with FK506-binding proteins (FKBPs); immunophilins, with cis-trans peptidyl-prolyl isomerase activity. FKBP12 copurifies with RyR1 (skeletal isoform) and modulates its gating. A different form of FKBP with a slightly higher molecular weight copurifies with RyR2 (cardiac isoform). Previous studies have demonstrated that FKBP stablizes gating of the skeletal Ca(2+)-release channel. In the present study, we measured the activity of cardiac RyRs incorporated into planar lipid bilayers to show that rapamycin, a drug that inhibits the prolyl isomerase activity of FKBP and dissociates FKBP from the RyR, increases the open probability and reduces the current amplitude of cardiac muscle Ca(2+)-release channels. These experiments show for the first time that submicromolar concentrations of rapamycin can alter channel function. Our results provide support for the hypotheses that FKBP functionally associates with the RyR and that the immunosuppressant drug, rapamycin, alters the function of both cardiac and skeletal muscle isoforms of the Ca(2+)-release channel. Our findings suggest that FKBP-dependent modulation of channel function may be generally applicable to all members of the intracellular Ca(2+)-release channel family and that FKBPs may play important regulatory roles in many cell processes, ranging from long-term depression in neurons to contractility in cardiomyocytes.