Axonal Kv1 channels regulate action potential propagation-an evolutionarily conserved function important for the control of motor behavior as evidenced from the linkage of human Kv1 channel mutations to myokymia/episodic ataxia type 1 (EA1) and the Shaker mutant phenotype in Drosophila. To search for the machinery that mediates axonal targeting of Kv1 channels composed of both alpha and beta subunits, we first demonstrate that Kvbeta2 is responsible for targeting Kv1 channels to the axon. Next, we show that Kvbeta2 axonal targeting depends on its ability to associate with the microtubule (MT) plus-end tracking protein (+TIP) EB1. Not only do Kvbeta2 and EB1 move in unison down the axon, Brefeldin A-sensitive Kv1-containing vesicles can also be found at microtubule ends near the cell membrane. In addition, we found that Kvbeta2 associates with KIF3/kinesin II as well. Indeed, Kv1 channels rely on both KIF3/kinesin II and EB1 for their axonal targeting.