The membrane-spanning S6 segments of the cardiac Na channel (Nav1.5) harbor amino acids that form the cytoplasmic entrance of the channel and are important determinants of gating and pharmacology. Despite their functional significance, the S6 amino acids that line the cytoplasmic pore of the Nav1.5 channel have not been clearly established. To identify residues exposed within the aqueous pore, cysteines were introduced into the S6 segment of the fourth homologous domain (DIVS6) and the mutant channels examined for sensitivity to a thiol-specific reagent (MTSET). Internally applied MTSET reduced the peak currents, induced hyperpolarizing shifts of steady-state availability, and slowed the recovery of the Y1767C and V1763C mutants. The MTSET inhibition of these cysteine mutants was voltage-dependent and well correlated with the steady-state availability of the MTSET-modified channels suggesting a link between inactivation and MTSET modification. The role of inactivation was further investigated by transferring the DIVS6 cysteine mutations to an inactivation-deficient background created by replacing a conserved phenylalanine (F1486) of the DIII-DIV linker with cysteine (ICM) or IQM. Internal MTSET abolished the inactivation of the V1763C-ICM and Y1767C-ICM mutants and attenuated the MTSET inhibition. MTSET modification of a cysteine introduced near the intracellular end of the DIVS6 segment (I1770C) disrupted fast inactivation suggesting that this region may contribute to a binding site for the inactivation gate. The data suggest that the docking of the inactivation gate near the cytoplasmic entrance of the channel induces a localized conformational change that regulates the aqueous accessibility of residues situated near the C-terminus of the DIVS6 segment. State-dependent changes in the pore lining have important implications for Na channel pharmacology.