The Ca²⁺-sensing receptor (CaR), a G protein-coupled receptor, is expressed in many epithelial tissues including the parathyroid glands, kidney, and GI tract. Although its role in regulating PTH levels and Ca²⁺ metabolism are best characterized, it may also regulate salt and water transport in the kidney. In order to determine if this receptor interacts with novel proteins that control ion transport, we screened a human adult kidney cDNA library with the C-terminal 219 amino acid cytoplasmic tail of the CaR as bait using the yeast two hybrid system. We identified two independent clones coding for approximately 125 aa from the C-terminus of the inwardly-rectifying K²⁺ channel, Kir4.2. The CaR and Kir4.2 as well as Kir4.1 (another member of Kir4 subfamily) were reciprocally co-immunoprecipitated from HEK-293 cells in which they were expressed, but the receptor did not co-immunoprecipitate with Kir5.1 or Kir1.1. Both Kir4.1 and Kir4.2 were immunoprecipitated from rat kidney extracts with the CaR. In Xenopus oocytes, expression of the CaR with either Kir4.1 or Kir4.2 channels resulted in inactivation of whole cell current as measured by two-electrode voltage clamp, but the non-functional CaR mutant CaR^R796W, and that does not co-immunoprecipitate with the channels, had no effect. Kir4.1 and the CaR were co-localized in the basolateral membrane of the distal nephron. The CaR interacts directly with Kir4.1 and Kir4.2 and can decrease their currents, which in turn could reduce recycling of K⁺ for the basolateral Na⁺, K⁺-ATPase and thereby contribute to inhibition of Na⁺ reabsorption.