Some blockers of voltage-gated Na+ and Ca2+ channels are assumed to pass through the membrane and then bind to amino acids in the internal vestibule by access from the internal side of the membrane. However, in the heart isoform of the voltage-gated Na+ channel, in L-type calcium channels and in T-type calcium channels an additional external access pathway (EAP) through the protein has been suggested. Furthermore, in voltage-gated Na+ channels (NaV) mutations at a specific site in the middle of the domain IV transmembrane segment 6 (site 1575 in rNaV1.4, 1760 in rNaV1.4) open an EAP for QX-222, a permanently charged, hydrophilic lidocaine analogue. Recently, the first crystal structure of a NaV was published . In this bacterial channel structure (NaVAb) the side chain homologous to rNaV1.4 I1575 (I202 in NaVAb) is in close contact with a pore-loop sidechain, homologous to rNaV1.4 W1531 (W179 in NaVAb). In contrast, in all currently available structural homology models of NaV, W1531 is not in contact with I1575. If W1531 were positioned as suggested in the NaVAb structure then a reduction in the length of the side chain at this site would be predicted to open the EAP. To test this hypothesis we generated the mutations W1531A and W1531G and tested these constructs for block by external QX-222.