Sphingosine-1-phosphate (S1P) is an endogenous agonist for a family of five G protein-coupled receptors (S1P(1-5)) involved in cell proliferation, cardiovascular development and lymphocyte trafficking. The sphingolipid drug FTY720 displays structural similarity to S1P and efficacy as an immunosuppressant in models of autoimmune disease and in solid organ transplantation. While FTY720 is well-tolerated in humans, it produces a transient reduction of heart rate (HR). As S1P activates the cardiac G protein-gated potassium channel I(KACh), we speculated that the FTY720-induced HR reduction reflects I(KACh) activation. We examined FTY720 effects on atrial myocytes from wild-type and I(KACh)-deficient mice. In wild-type myocytes, the active phosphate metabolite of FTY720 (FTY720-P) induced single channel activity with conductance, open time, GTP sensitivity and rectification identical to that of I(KACh). In whole-cell recordings, FTY720-P evoked an inwardly rectifying potassium current in approximately 90% of myocytes responding to acetylcholine. Comparable channel activity was never observed in myocytes from I(KACh)-deficient mice. In wild-type mice, acute FTY720 administration produced a dose-dependent, robust HR reduction. In contrast, the HR reduction induced by FTY720 in I(KACh)-deficient mice was blunted. We conclude that the effect of acute FTY720 administration on HR is mediated primarily by I(KACh) activation.