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 40x to 231x more effective, when compared to 4,9-ah-TTX, on a given isoform. An exception was Na_v1.6, where 4,9-ah-TTX in nmole/L 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 (in nmole/L): 7,8±1,3/1260±121 (Na_v1.2), 2,8±2,3/341±36 (Na_v1.3), 4,5±1,0/988 ±62 (Na_v1.4), 1970±565/78500 ±11600 (Na_v1.5), 3,8±1,5/7,8 ±2,3 (Na_v1.6), 5,5±1,4/1270±251 (Na_v1.7) and 1330±459/>30000 (Na_v1.8). Analysis of approximal half-maximal doses of both compounds revealed minor effects on voltage dependent activation only, while 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 ionchannels is frequently observed in the case of proteinaceous toxins. Although the sensitivity of Na_v1.1 to 4,9-anhydro-TTX is not known, here we report evidence on a highly isoform specific TTX analogue, 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.