The a subunit of voltage-dependent sodium channels comprises 4 domains, each of which with 6 transmembrane segments named S1-S6. Segments S4 of all domains contain positively charged amino acids that confer voltage-sensitivity to the channel. It has been shown that a-scorpion toxins bind to the site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. The binding occurs in a voltage-dependent way, decreasing at strong depolarized potentials. In order to investigate the movement of S4 segments in the presence of bound toxin, we have used Ts3, a a-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared to control conditions. Site-specific fluorescence measurements were carried out by labeling mutant channels, with cysteine introduced in the S3-S4 linker of each of the domains of the channel, with tetramethylrhodamine meleimide, one at a time. Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. It has been proposed previously that the movement of the voltage sensors in the sodium channels is tightly coupled. In order to verify if the bound Ts3 affects the movement of the other voltage sensors we measured the fluorescent signals produced by the movement of the first three voltage sensors. The fluorescence-voltage (F-V) relationships of domains II and III remained unaffected in the presence of Ts3. However, the toxin shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results support the proposed model, in which Ts3 impairs the movement of the S4-DIV to its activated position, but suggest the occurrence of a small movement of  the segment in relation to its surroundings.