Objective: Decay kinetics of the voltage-gated L-type Ca²⁺ current (I_CaL) control the magnitude of Ca²⁺ influx during the cardiac action potential. We investigated the influence of changes in diastolic membrane potential on I_CaL decay kinetics in cardiac cells. Methods: Cells were isolated enzymatically from rat ventricles, human right atrial appendages obtained during corrective heart surgery and left ventricles from end-stage failing hearts of transplant recipients. The whole-cell patch-clamp technique was used to evoke I_CaL by a 100-ms depolarizing test pulse to −10 mV. Conditioning potentials between −80 and 0 mV were applied for 5 s prior to the test pulse. Results: Depolarizing the cells between −80 and −50 mV prior to the test pulse slowed the early inactivation of I_CaL both in rat ventricular and human atrial cells. This slowing resulted in a significant increase of Ca²⁺ influx. This type of facilitation was not observed when the sarcoplasmic reticulum (SR) Ca²⁺ content was depleted using ryanodine which reduced the rate of inactivation of I_CaL, or when Ba²⁺ replaced Ca²⁺ as the permeating ion. Facilitation was favored by intracellular cAMP-promoting agents that, in addition to increasing current peak amplitude, enhanced the fast Ca²⁺-dependent inactivation of I_CaL. Facilitation was impaired in atrial and ventricular human failing hearts. Conclusion: Decay kinetics of I_CaL are regulated by the diastolic membrane potential in rat and human cardiomyocytes. This regulation, which associates slowing of I_CaL inactivation with reduced SR Ca²⁺ release and underlies facilitation of Ca²⁺ channels activity, may have profound physiological relevance for catecholamines enhancement of Ca²⁺ influx. It is impaired in failing hearts, possibly due to lowered SR Ca²⁺ release.