Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNa V1.4 F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNa V1.4 F1705I are incompletely understood. The location of the hNa V1.4 F1705I in the CT prompted us to examine the role of Ca 2+ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human Na V1.4 channel regulation by Ca 2+ and CaM. hNa V1.4 F1705I inactivation gating is Ca 2+-sensitive compared to wild type hNa V1.4 which is Ca 2+ insensitive and the mutant channel exhibits a depolarizing shift of the V 1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNa V1.4 channel background (rNa V1.4 F1698I) eliminates Ca 2+ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca 2+ sensitivity of gating between wild type and mutant human and rat Na V1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca 2+/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows I Na decay in a Ca 2+-sensitive fashion. The combination of the altered voltage dependence and kinetics of I Na decay contribute to the myotonic phenotype and may involve the Ca 2+-sensing apparatus in the CT of Na V1.4.