The blocking efficacy of 4,9-anhydro-TTX (4,9-ah-TTX) and TTX on several isoforms of voltage-dependent sodium channels, expressed in Xenopus laevis oocytes, was tested (Na_v1.2, Na_v1.3, Na_v1.4, Na_v1.5, Na_v1.6, Na_v1.7, and Na_v1.8). Generally, TTX was 40–231 times more effective, when compared with 4,9-ah-TTX, on a given isoform. An exception was Na_v1.6, where 4,9-ah-TTX in nanomole per liter concentrations sufficed to result in substantial block, indicating that 4,9-ah-TTX acts specifically at this peculiar isoform. The IC₅₀ values for TTX/4,9-ah-TTX were as follows (in nmol/l): 7.8 ± 1.3/1,260 ± 121 (Na_v1.2), 2.8 ± 2.3/341 ± 36 (Na_v1.3), 4.5 ± 1.0/988 ± 62 (Na_v1.4), 1,970 ± 565/78,500 ± 11,600 (Na_v1.5), 3.8 ± 1.5/7.8 ± 2.3 (Na_v1.6), 5.5 ± 1.4/1,270 ± 251 (Na_v1.7), and 1,330 ± 459/>30,000 (Na_v1.8). Analysis of approximal half-maximal doses of both compounds revealed minor effects on voltage-dependent activation only, whereas steady-state inactivation was shifted to more negative potentials by both TTX and 4,9-ah-TTX in the case of the Na_v1.6 subunit, but not in the case of other TTX-sensitive ones. TTX shifted steady-state inactivation also to more negative potentials in case of the TTX-insensitive Na_v1.5 subunit, where it also exerted profound effects on the time course of recovery from inactivation. Isoform-specific interaction of toxins with ion channels is frequently observed in the case of proteinaceous toxins. Although the sensitivity of Na_v1.1 to 4,9-ah-TTX is not known, here we report evidence on a highly isoform-specific TTX analog that may well turn out to be an invaluable tool in research for the identification of Na_v1.6-mediated function, but also for therapeutic intervention.