Voltage-gated potassium (K+) channels are assembled by four identical or homologous alpha-subunits to form a tetrameric complex with a central conduction pore for potassium ions. Most of the cloned genes for the alpha-subunits are classified into four subfamilies: Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw), and Kv4 (Shal). Subfamily-specific assembly of heteromeric K+ channel complexes has been observed in vitro and in vivo, which contributes to the diversity of K+ currents. However, the molecular codes that mediate the subfamily-specific association remain unknown. To understand the molecular basis of the subfamily-specific assembly, we tested the protein-protein interactions of different regions of alpha-subunits. We report here that the cytoplasmic NH2-terminal domains of Kv1, Kv2, Kv3, and Kv4 subfamilies each associate to form homomultimers. Using the yeast two-hybrid system and eight K+ channel genes, two genes (one isolated from rat and one from Drosophila) from each subfamily, we demonstrated that the associations to form heteromultimers by the NH2-terminal domains are strictly subfamily-specific. These subfamily-specific associations suggest a molecular basis for the selective formation of heteromultimeric channels in vivo.