The isoform specific role of sialic acid in human voltage-gated sodium channel gating was investigated through expression and chimeric analysis of two human isoforms, Na(v1.4) (hSkM1), and Na(v1.5) (hH1) in Chinese hamster ovary (CHO) cell lines. Immunoblot analyses indicate that both hSkM1 and hH1 are glycosylated and that hSkM1 is more glycosylated than hH1. Four sets of voltage-dependent parameters, the voltage of half-activation (V(a)), the voltage of half-inactivation (V(i)), the time constants for fast inactivation (tau(h)), and the time constants for recovery from inactivation (tau(rec)), were measured for hSkM1 and hH1 expressed in two CHO cell lines, Pro5 and Lec2, to determine the effect of changing sialylation on channel gating under conditions of full (Pro5) or reduced (Lec2) sialylation. For all parameters measured, hSkM1 gating showed a consistent 11-15 mV depolarizing shift under conditions of reduced sialylation, while hH1 showed no significant change in any gating parameter. Shifts in channel V(a) with changing external [Ca2+] indicated that sialylation of hSkM1, but not hH1, directly contributes to a negative surface potential. Functional analysis of two chimeras, hSkM1P1 and hH1P1, indicated that the responsible sialic acids are localized to the hSkM1 S5-S6 loop of domain I. When hSkM1 IS5-S6 was replaced by the analogous hH1 loop (hSkM1P1), changing sialylation had no significant effect on any voltage-dependent parameter. Conversely, when hSkM1 IS5-S6 was added to hH1 (hH1P1), all four parameters shifted by 6-7 mV in the depolarized direction under conditions of reduced sialylation. In summary, the gating of two human sodium channel isoforms show very different dependencies on sialic acid, with hSkM1 gating uniformly altered by sialic acid levels through an apparent electrostatic mechanism, while hH1 gating is unaffected by changing sialylation. Sialic acid-dependent gating can be removed or created by replacing or inserting hSkM1 IS5-S6, respectively, indicating that the functionally relevant sialic acid residues are localized to the first domain of the channel.