Ca²⁺ current (I_Ca) recovery from inactivation is necessary for normal cardiac excitation-contraction coupling. In normal hearts increased stimulation frequency increases force, but in heart failure (HF) this force-frequency relationship (FFR) is often flattened or reversed. While reduced SR Ca²⁺ ATPase function may be involved, decreased I_Ca availability may also contribute. Longer action potential duration (APD), slower [Ca²⁺]_i decline and higher diastolic [Ca²⁺]_i in HF could all slow I_Ca recovery from inactivation, thereby decreasing I_Ca availability. We measured the effect of different diastolic [Ca²⁺]_i on I_Ca inactivation and recovery from inactivation in rabbit cardiac myocytes. Both I_Ca and Ba²⁺ current (I_Ba) were measured. I_Ca decay was accelerated only at high diastolic [Ca²⁺]_i (600 nM). I_Ba inactivation was slower, but insensitive to [Ca²⁺]_i. V_m-dependence of I_Ca or I_Ba availability was not affected by [Ca²⁺]_i below 600 nM. Recovery from inactivation was slowed by both depolarization and high [Ca²⁺]_i. We also used perforated patch with AP-clamp and normal [Ca²⁺]_i transients, using various APDs as conditioning pulses for different frequencies (and to simulate HF APD). Recovery of I_Ca following longer APD was increasingly incomplete, decreasing I_Ca availability. Trains of long APs caused a larger I_Ca decrease than short APD at the same frequency. This effect on I_Ca availability was exacerbated by slowing twitch [Ca²⁺]_i decline by ~50%. We conclude that long APD and slower [Ca²⁺]_i decline leads to cumulative inactivation limiting I_Ca at high heart rates and might contribute to the negative FFR in HF, independent of altered Ca²⁺ channel properties.