Aim: Several β-subunits have been suggested to modulate the electrophysiologi-cal properties of the transient outward current (Ito) in cardiac myocytes, includ-ing the obligatory β-subunit K+-channel interacting protein (KChIP2) and KCNE2. However, neither KChIP2 nor KCNE2 modulation of Kv4.x (x=2 and/or 3) can fully recapitulate the electrophysiological properties of native Ito. The present study is to investigate how Ito current is modulated when both KChIP2 and KCNE2 are co-expressed. Methods: Kv4.2, KChIP2c, and KCNE2 cDNA were simultaneously transfected into COS-7 cells at a molar ratio of 3:1:1. Whole-cell currents were recorded by the patch-clamp method. Results: In comparison with the current regulated by KChIP2c alone, the co-expression of KCNE2 further slowed Kv4.2 current inactivation kinetics, but diminished KChIP2c-induced positive shift of the voltage-dependent activation of Kv4.2 current. Importantly, co-expression of KCNE2 accelerated the current recovery from inactivation, and caused an "over-shoot" of peak current amplitude during Kv4.2 current recovery, a phenomenon which has been uniquely described for human Ito. However, co-expression of KCNE2 exerted no further effect on Kv4.2 current amplitude, the rate of Kv4.2 current activation and voltage-dependent inactivation. Conclusion: Co-expres-sion of Kv4.2 with KChIP2c and KCNE2, but not with KChIP2c or KCNE2 alone, yields a current profile similar to native Ito. Both KChIP2c and KCNE2 simulta-neously participate in recapitulation of the electrophysiological properties of Ito in cardiac myocytes.