Three different experimental approaches were used to establish that the first transmembrane segment (S1) is important for K+ channel assembly. First, hydrodynamic analyses of in vitro translated Kv1.1 N-terminal domain containing the S1 segment coassembles to form homotetrameric complexes, whereas deletion of the S1 segment abolishes coassembly. Second, coimmunoprecipitation experiments of cotranslated Kv1.1 and Kv1.5 truncated polypeptides show that the S1 segment is essential for coimmunoprecipitation. Third, dominant negative experiments in Xenopus oocytes confirm that over-expression of either the S1 segment or the N-terminal domain is sufficient for abolishing the expression of functional Kv1.1 and Kv1.5 K+ channels. These data indicate that S1 segment plays an important role in the coassembly of homo- and heterotetrameric K+ channels.