Kv4 channels are differentially expressed across the mouse left ventricular free wall. Accordingly, the transient outward K + current ( I to ), which is produced by Kv4 channels, is greater in left ventricular epicardial (EPI) than in endocardial (ENDO) cells. However, the mechanisms underlying heterogeneous Kv4 expression in the heart are unclear. Here, we tested the hypothesis that differential [Ca 2+ ] i and calcineurin/NFATc3 signaling in EPI and ENDO cells contributes to the gradient of I to function in the mouse left ventricle. In support of this hypothesis, we found that [Ca 2+ ] i , calcineurin, and NFAT activity were greater in ENDO than in EPI myocytes. However, the amplitude of I to was the same in ENDO and EPI cells when [Ca 2+ ] i , calcineurin, and NFAT activity were equalized. Consistent with this, we observed complete loss of I to and Kv4 heterogeneity in NFATc3-null mice. Interestingly, Kv4.3, Kv4.2, and KChIP2 genes had different apparent thresholds for NFATc3-dependent suppression and were ordered as Kv4.3≈KChIP2>Kv4.2. Based on these data, we conclude that calcineurin and NFATc3 constitute a Ca 2+ -driven signaling module that contributes to the nonuniform distribution of Kv4 expression, and hence I to function, in the mouse left ventricle.