In this report we demonstrate that in HEK293 cells stably expressing the human V2 vasopressin receptor, ligand-induced internalization of the hormone receptor occurs via the clathrin-dependent pathway. Studies of receptor trafficking either by direct visualization of the V2 receptor by confocal microscopy or binding experiments show a rapid internalization (half-time 6-7 min). Blocking of the clathrin-dependent pathway by hypertonic sucrose increased vasopressin-induced cellular cAMP production and decreased the desensitization of the V2 receptor-adenylyl cyclase system. Thus, internalization appears to be a major regulatory mechanism terminating vasopressin action in HEK293 cells. Two antagonists of the vasopressin V2 receptor exerted different effects on receptor internalization, as determined by confocal fluorescence microscopy. The nonpeptidic antagonist OPC31260 did not induce any visible receptor internalization, whereas the peptidic antagonist d(CH2)5[D-Tyr(Et)2,Val4,Lys8,Tyr-NH29]VP induced a slow but substantial receptor internalization. These results suggest that long-term treatment with peptidic V2 receptor antagonists might lead to desensitization.