FK506, a widely used immunosuppressant, has caused a few clinical cases with QT prolongation and torsades de pointe at high blood concentration. The proarrhytmogenic potential of FK506 was investigated in single rat ventricular cells using the whole-cell clamp method to record action potentials (APs) and ionic currents. Fluorescence measurements of Ca²⁺ transients were performed with Indo-1 AM using a multiphotonic microscope. FK506 (25 µmol/L) hyperpolarized the resting membrane potential (RMP, -3 mV) and prolonged APs (APD₉₀ increased by 21%) at 0.1 Hz. Prolongation was enhanced by three-fold at 3.3 Hz and early afterdepolarizations (EADs) occurred in 59% of cells. EADs were prevented by stronger intracellular Ca²⁺ buffering (EGTA: 10 vs 0.5 mmol/L in the patch-pipette), or replacement of extracellular Na⁺ by Li⁺ which abolishes Na⁺-Ca²⁺ exchange (I_Na-Ca). In Indo-1-loaded cells, FK506 generated doublets of Ca²⁺ transients associated with increased diastolic Ca²⁺ in half of the cells. FK506 reversibly decreased the L-type Ca²⁺ current (I_CaL) by 25%, although high-frequency dependent facilitation of I_CaL persisted, and decreased three distinct K⁺ currents: I_K (>80%), I_to (<20%), and I_K1 (>40%). A shift in the reversal potential of I_K1 (-5 mV) accounted for RMP hyperpolarization. Numerical simulations, reproducing all experimental effects of FK506, and use of nifedipine showed that frequency-dependent facilitation of I_CaL plays a role in the occurrence of EADs. In conclusion, effects FK506 on the cardiac AP are more complex than previously reported and include inhibitions of I_K1 and I_CaL. Alterations in Ca²⁺ release and I_Na-Ca may contribute to FK506-induced AP prolongation and EADs, in addition to the permissive role of I_CaL facilitation at high rates of stimulation.