FK-506 binding protein (FKBP) has been reported to be closely associated with the ryanodine receptor in skeletal and cardiac muscle and to modulate sarcoplasmic reticulum (SR) Ca²⁺ release channel gating in isolated channels. FK-506 can inhibit the activity of FKBP, thereby reversing its effects on SR Ca²⁺ release. We investigated the function of FKBP during normal contractions and Ca²⁺ transients in intact rat ventricular myocytes loaded with fluorescent Ca²⁺ indicators. FK-506 significantly increased steady state twitch Ca²⁺ transients and contraction amplitudes even under conditions in which the SR Ca²⁺ load and Ca²⁺ current were unaltered, suggesting that FK-506 increases the fraction of SR Ca²⁺ released during excitation-contraction (E-C) coupling. Action potentials were somewhat prolonged, consistent with the larger Ca²⁺ transients causing greater inward Na⁺-Ca²⁺ exchange current. FK-506 did not affect SR Ca²⁺ uptake but modestly decreased Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange in intact cells (although no effect on Na⁺-Ca²⁺ exchange was seen in sarcolemmal vesicles). In most cells, FK-506 caused an increase in SR Ca²⁺ content during steady state stimulation, as assessed by caffeine-induced contractures. This was probably due to the inhibition of Ca²⁺ efflux via Na⁺-Ca²⁺ exchange. FK-506 also accelerated the rest decay of SR Ca²⁺ content and increased the frequency of resting Ca²⁺ sparks about fourfold. The increase in frequency of these basic Ca²⁺ release events was not associated with changes in the amplitude or duration of the Ca²⁺ sparks. We conclude that FK-506 increases the fraction of SR Ca²⁺ released during normal twitches and enhances the rate of SR Ca²⁺ release during rest. FK-506 also inhibits Na⁺-Ca²⁺ exchange, although this effect may be indirect. These effects are consistent with an important SR-stabilizing effect of FKBP in intact rat ventricular myocytes.