-Spectrin and ankyrin are major components of the membrane cytoskeleton. We have generated mice carrying a null mutation in the IV-spectrin gene using gene trapping in embryonic stem cells. Mice homozygous for the mutation exhibit tremors and contraction of hindlimbs. IV-spectrin expression is mostly restricted to neurons, where it colocalizes with and binds to ankyrin-G at axon initial segments (AISs) and nodes of Ranvier (NR). In IV-spectrin–null neurons, neither ankyrin-G nor voltage-gated sodium channels (VGSC) are correctly clustered at these sites, suggesting that impaired action potential caused by mislocalization of VGSC leads to the phenotype. Conversely, in ankyrin-G–null neurons, IV-spectrin is not localized to these sites. These results indicate that IV-spectrin and ankyrin-G mutually stabilize the membrane protein cluster and the linked membrane cytoskeleton at AIS and NR.