Complex modulation of voltage-gated Ca2+ currents through the interplay among Ca2+ channels and various Ca(2+)-binding proteins is increasingly being recognized. The K+ channel interacting protein 2 (KChIP2), originally identified as an auxiliary subunit for K(V)4.2 and a component of the transient outward K+ channel (I(to)), is a Ca(2+)-binding protein whose regulatory functions do not appear restricted to K(V)4.2. Consequently, we hypothesized that KChIP2 is a direct regulator of the cardiac L-type Ca2+ current (I(Ca,L)). We found that I(Ca,L) density from KChIP2(-/-) myocytes is reduced by 28% compared to I(Ca,L) recorded from wild-type myocytes (P<0.05). This reduction in current density results from loss of a direct effect on the Ca2+ channel current, as shown in a transfected cell line devoid of confounding cardiac ion currents. I(Ca,L) regulation by KChIP2 was independent of Ca2+ binding to KChIP2. Biochemical analysis suggested a direct interaction between KChIP2 and the Ca(V)1.2 alpha(1C) subunit N terminus. We found that KChIP2 binds to the N-terminal inhibitory module of alpha(1C) and augments I(Ca,L) current density without increasing Ca(V)1.2 protein expression or trafficking to the plasma membrane. We propose a model in which KChIP2 impedes the N-terminal inhibitory module of Ca(V)1.2, resulting in increased I(Ca,L). In the context of recent reports that KChIP2 modulates multiple K(V) and Na(V) currents, these results suggest that KChIP2 is a multimodal regulator of cardiac ionic currents.