To prove heteromeric assembly of KChIP proteins, the present study is carried out. The results of chemical crosslinking and pull down assay revealed that KChIP1, KChIP2.1, and KChIP2.2 could form homo- as well as hetero-oligomer, and this oligomerization exhibited a Ca(2+)-dependent manner. Moreover, homomeric and heteromeric assembly of KChIPs did not perturb their interaction with Kv4.2 K(+) channel, indicating that the region associated with oligomerization of KChIPs was distinct from that for binding with Kv4.2. Together with previous findings that the net effects of KChIP proteins on the molecular properties and trafficking of Kv channel were different, these observations open a fascinating possibility that the electrophysiological properties of Kv channel may be differently regulated by homomeric and heteromeric assembly of KChIPs.