FK-506 increases the cytosolic Ca2+concentration transient in rat ventricular myocytes by prolonging the action potential through inhibition of the K+currents Itoand IK[ J. Physiol. (Lond.) 501: 509–516, 1997]. Physiological and biochemical techniques were used in parallel to examine the electrophysiological mechanisms and the role of calcineurin inhibition in these effects. FK-506 prolonged the recovery of Itofrom inactivation. Thus Itoinhibition was frequency dependent, with no decrease at 0.2 Hz (recorded at +50 mV from −70 mV) but a 40% decrease at 2.0 Hz. In contrast, inhibition of IK(∼60%) was time and voltage independent. At 25 μM, FK-506 (by 65%) and cyclosporin A (by 57%) inhibited calcineurin activity in myocyte extracts. However, only FK-506 increased the cytosolic Ca2+concentration transient in field-stimulated myocytes. Furthermore, FK-506 was still active on K+currents when cells were dialyzed with 10 mM EGTA. These results demonstrate that calcineurin inhibition is not responsible for the functional effects of FK-506 in heart and suggest that IKand Itoare modulated by FK-506-binding proteins or directly by FK-506.