A transient outward K⁺ current (I_to) mediates the initial repolarization in ventricular myocytes, thereby setting the membrane potential for the action potential plateau phase, which is carried by Ca²⁺ influx. Cytoplasmic Ca²⁺ controls contraction of the myofilaments; but also, the function of many signalling proteins in the plasma membrane, including ion channels, may be modulated indirectly or directly by cytoplasmic Ca²⁺. A molecular substrate of I_to in the human heart is the Kv4.3 channel, associated with Kv Channel Interacting Protein (KChIP) 2. These accessory KChIP2 subunits modulate Kv4.3 channel gating by slowing macroscopic inactivation and accelerating recovery from inactivation. Notably, KChIPs are Ca²⁺-binding proteins with three functional EF-hand motifs (EF2, EF3 and EF4). We asked if binding of Ca²⁺ to these EF-hand motifs directly influences the inactivation behaviour of Kv4.3/KChIP2 complexes. To answer this question we expressed Kv4.3 channels together with wild-type and EF-hand-mutated KChIP2 in HEK 293 cells by transient transfection. After 18 – 48 h we performed whole-cell patch-clamp experiments with different Ca²⁺ concentrations in the patch pipette. We found that, unlike the onset of macroscopic inactivation, the recovery of Kv4.3/KChIP2 channel complexes from inactivation was modulated in a Ca²⁺-dependent manner. Kv4.3 recovery from inactivation was always accelerated by KChIP2 coexpression, but proceded slower with 50 M internal free Ca²⁺ (t50 = 148 ms) and faster after BAPTA-AM pretreatment and BAPTA in the patch pipette (tBAPTA = 66 ms; p<0.001). Accelerated Kv4.3 recovery kinetics but no Ca²⁺ effect on recovery kinetics was observed when Kv4.3 was coexpressed with the mutant KChIP2DEF2 (t50 = 105 ms, tBAPTA = 104 ms; p=0.98). These data suggest a pivotal role of Ca²⁺ binding to EF2 in KChIP2, which is unrelated to Kv4.3/KChIP2 assembly.