ROMK channels are present in the cortical collecting ducts of kidney and are responsible for K⁺secretion in this nephron segment. Recent studies suggest that endocytosis of ROMK channels is important for regulation of K⁺ secretion in cortical collecting ducts. We investigated the molecular mechanisms for endocytosis of ROMK channels expressed inXenopus laevis oocytes and cultured Madin-Darby canine kidney cells. When plasma membrane insertion of newly synthesized channel proteins was blocked by incubation with brefeldin A, ROMK currents decreased with a half-time of ∼6 h. Coexpression with the Lys44→Ala dominant-negative mutant dynamin, but not wild-type dynamin, reduced the rate of reduction of ROMK in the presence of brefeldin A. Mutation of Asn371 to Ile in the putative NPXY internalization motif of ROMK1 abolished the effect of the Lys44→Ala dynamin mutant on endocytosis of the channel. Coimmunoprecipitation study and confocal fluorescent imaging revealed that ROMK channels associated with clathrin coat proteins in Madin-Darby canine kidney cells. These results provide compelling evidence for endocytosis of ROMK channels via clathrin-coated vesicles.