In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K⁺ current (I_to) by calmodulin kinase II (CamKII). Intracellular application of CamKII inhibitors KN93, calmidazolium and autocamtide-2 related inhibitory peptide II (ARIP-II) accelerated the inactivation of I_to, even at low [Ca²⁺]. In the same conditions, CamKII coimmunoprecipitated with Kv4.3 channels suggesting that phosphorylation of Kv4.3 channels modulate inactivation of I_to. As channels underlying Ito are heteromultimers of Kv4.2 and Kv4.3 we have explored the effect of CamKII on HEK cells transfected with either of those Kv subunits. While Kv4.3 inactivated faster upon inhibition of CamKII, Kv4.2 inactivation was insensitive to CamKII inhibitors. However, Kv4.2 inactivation became slower when high Ca²⁺ was used in the pipette or when [Ca²⁺]_i was transiently increased. This effect was inhibited by KN93, and western blot analysis demonstrated Ca²⁺ dependent phosphorylation of Kv4.2 channels. On the contrary, CamKII coimmunoprecipitated with Kv4.3 channels without a previous Ca²⁺ increase, and the association was inhibited by KN93. These results suggest that both channels underlying I_to are substrates of CamKII although with different sensitivities: Kv4.2 remain unphosphorylated unless [Ca²⁺]_i increases, while Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CamKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CamKII in the membrane, allowing Ca²⁺ dependent modulation by this enzyme of the associated Kv4.2 channels.