In voltage-gated Na channels several amino acids of the S6 segment of domain IV (DIV-S6) have an important role in gating and local anesthetic drug block. Accordingly, mutations at several sites of this region have been shown to substantially alter the channel's inactivation properties. For a comprehensive analysis of the kinetic role of DIV-S6 in fast inactivation we performed a cysteine scanning mutagenesis of sites 1575–1591 in the DIV-S6 of the rNav1.4 channel. In addition, we produced the same cysteine replacements in the background of the mutation in the putative selectivity filter K1237E. K1237 is located in the P-loop of domain III and homology modelling based on the crystal structure of the KcsA channel suggests a close spacial relationship between K1237 and I1575 of the DIV-S6 segment. The mutation K1237E dramatically alters both the permeation properties and the gating behavior of the channel. Hence, this mutation most likely causes a complex conformational rearrangement of the channel. We sought to investigate whether K1237E changes the pattern of the gating perturbations produced by the serial cysteine replacements in DIV-S6. All constructs were expressed in Xenopus laevis oocytes and studied by means of two electrode voltage-clamp. The half-point of availability following a 50 ms conditioning prepulse (V05) was -44 1 mV and -51 1 mV in wild-type and K1237E, respectively (P < 0.001) . Most serial amino acid replacements by cysteines in DIV-S6 produced shifts in V05, both in the background of wild-type and in the background of K1237E, ranging from +17 1 mV to -9 2 mV. A plot of the shifts in V05 by single DIV-S6 mutants relative to wild-type versus the shifts in V05 by double mutants relative to K1237E showed a significant positive correlation (R= 0.72, P=0.002). This indicates that the general pattern of gating perturbations in DIV-S6 is not affected by K1237E, suggesting a high conformational stability of the DIV-S6 segment during the fast inactivated state.