High densities of ion channels at axon initial segments (AISs) and nodes of Ranvier are required for initiation, propagation, and modulation of action potentials in axons. The organization of these membrane domains depends on a specialized cytoskeleton consisting of two submembranous cytoskeletal and scaffolding proteins, ankyrinG (ankG) and βIV spectrin. However, it is not known which of these proteins is the principal organizer, or if the mechanisms governing formation of the cytoskeleton at the AIS also apply to nodes. We identify a distinct protein domain in βIV spectrin required for its localization to the AIS, and show that this domain mediates βIV spectrin's interaction with ankG. Dominant-negative ankG disrupts βIV spectrin localization, but does not alter endogenous ankG or Na⁺ channel clustering at the AIS. Finally, using adenovirus for transgene delivery into myelinated neurons, we demonstrate that βIV spectrin recruitment to nodes of Ranvier also depends on binding to ankG.