Prokaryotic voltage-gated sodium channels (Na(V)s) are homotetramers and are thought to inactivate through a single mechanism, named C-type inactivation. Here we report the voltage dependence and inactivation rate of the NaChBac channel from Bacillus halodurans, the first identified prokaryotic Na(V), as well as of three new homologues cloned from Bacillus licheniformis (Na(V)BacL), Shewanella putrefaciens (Na(V)SheP), and Roseobacter denitrificans (Na(V)RosD). We found that, although activated by a lower membrane potential, Na(V)BacL inactivates as slowly as NaChBac. Na(V)SheP and Na(V)RosD inactivate faster than NaChBac. Mutational analysis of helix S6 showed that residues corresponding to the "glycine hinge" and "PXP motif" in voltage-gated potassium channels are not obligatory for channel gating in these prokaryotic Na(V)s, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in Na(V)BacL (A214G), Na(V)SheP (A216G), and NaChBac (G219A) accelerated inactivation in these channels, whereas mutation of glycine to alanine in the lower part of helix S6 in NaChBac (G229A), Na(V)BacL (G224A), and Na(V)RosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic Na(V)s does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels.