Interactions with ankyrinG are crucial to the localization of voltage-gated sodium channels (VGSCs) at the axon initial segment and for neurons to initiate action potentials. However, the molecular nature of these interactions remains unclear. Here we report that VGSC-alpha, but not -beta, subunits bind to ankyrinG using pull-down assays. Further dissection of this activity identifies a conserved 9-amino acid motif ((V/A)P(I/L)AXXE(S/D)D) required for ankyrinG binding. This motif is also required for the localization of chimeric neurofascin/sodium channel molecules to the initial segment of cultured hippocampal neurons. The conserved nature of this motif suggests that it functions to localize sodium channels to a variety of "excitable" membrane domains both inside and outside of the nervous system.