K+ channel function is regulated through the assembly of channel subunit isoforms into either homo- or heterotetrameric structures each characterized by distinct pharmacologic and kinetic properties. In studying the molecular basis of subunit association in mammalian Shaker-like K+ channels, we constructed deletion mutants of the inactivating K+ channel hKv1.4 alone and in tandem with hKv1.5 and examined the functional properties electrophysiologically in Xenopus oocytes. Deletion of 255 amino acids in the amino-terminal domain of hKv1.4 prevented the formation of hybrid channels within the subfamily but had no effect on homomultimerization or voltage-dependent gating. The amino-terminal deletion mutant of Kv2.1, a noninactivating K+ channel from a distantly related subfamily also forms functional homomultimeric channels. Although members of different K+ channel subfamilies do not coassemble, coexpression of the amino-terminal deletion mutants of hKv1.4 and Kv2.1 resulted in the formation of functional hybrid channels. These results demonstrate that the amino-terminal region of mammalian K+ channels subserves two functions. It provides a recognition site necessary for hetero- but not homomultimeric channel assembly within a subfamily and prevents coassembly between subfamilies.