Strong inward rectifier potassium (Kir2) channels are important in the control of cell excitability, and their functions are modulated by interactions with intracellular proteins. Here we identified a complex of scaffolding/trafficking proteins in brain that associate with Kir2.1, Kir2.2, and Kir2.3 channels. By using a combination of affinity interaction pulldown assays and co-immunoprecipitations from brain and transfected cells, we demonstrated that a complex composed of SAP97, CASK, Veli, and Mint1 associates with Kir2 channels via the C-terminal PDZ-binding motif. We further demonstrated by using in vitro protein interaction assays that SAP97, Veli-1, or Veli-3 binds directly to the Kir2.2 C terminus and recruits CASK. Co-immunoprecipitations indicated that specific Veli isoforms participate in forming distinct protein complexes in brain, where Veli-1 stably associates with CASK and SAP97, Veli-2 associates with CASK and Mint1, and Veli-3 associates with CASK, SAP97, and Mint1. Additionally, immunocytochemistry of rat cerebellum revealed overlapping expression of Kir2.2, SAP97, CASK, Mint1, with Veli-1 in the granule cell layer and Veli-3 in the molecular layer. We propose a model whereby Kir2.2 associates with distinct SAP97-CASK-Veli-Mint1 complexes. In one complex, SAP97 interacts directly with the Kir2 channels and recruits CASK, Veli, and Mint1. Alternatively, Veli-1 or Veli-3 interacts directly with the Kir2 channels and recruits CASK and SAP97; association of Mint1 with the complex requires Veli-3. Expression of Kir2.2 in polarized epithelial cells resulted in targeting of the channels to the basolateral membrane and co-localization with SAP97 and CASK, whereas a dominant interfering form of CASK caused the channels to mislocalize. Therefore, CASK appears to be a central protein of a macromolecular complex that participates in trafficking and plasma membrane localization of Kir2 channels.