There are at least five subfamilies of Shaker-like K+ channels. The diverse function of K+ channels are thought to be further modulated by hydrophilic beta subunits. Here we report that Kv beta 1 inactivates RCK4 and Shaker B K+ channels of the Kv1 subfamily, but not Shal2 of the Kv4 subfamily. This correlates the subfamily-specific bindings of Kv beta 1 to the cytoplasmic N-terminal domains of Kv1 alpha subunits. We map the Kv beta 1-binding site to a region overlapping NABKv1, a domain that specifies different Kv1 alpha subunits to form heterotetramers. Using chimeric alpha subunits, we demonstrate that NABKv1 is essential for the Kv beta 1-mediated inactivation. These results suggest that Kv beta 1 modulates a subset of K+ channels through the specific assembly of alpha-beta complexes and reveal the dual function of the NAB domain in mediating the assembly of both alpha-alpha and alpha-beta complexes.