Receptor-mediated endocytosis has been observed after agonist occupation of several G protein-coupled receptors, which contributes to the desensitization response to agonist stimulation; however, the cellular signals required to initiate this process are unclear. In this study, we developed and characterized a new antagonist analogue of cholecystokinin (D-Tyr-Gly-[(Nle28,31,D-Trp30)cholecystokinin-26-32]-phen eth yl ester) that can be tagged with a fluorescent rhodamine and radioiodinated. This has permitted us to demonstrate that antagonist occupation of the cholecystokinin receptor also results in receptor internalization, which dissociates this response from second messenger signaling activities and receptor phosphorylation. Immunolocalization of this receptor after occupation with an established nonpeptidyl antagonist confirmed this phenomenon. Antagonist-induced receptor internalization probably results from stabilization of the receptor in a conformation that exposes a domain critical to directing it into the clathrin-dependent endocytic pathway. This work provides evidence for a new and independent mechanism for receptor internalization, provides a mechanism for the rarely observed phenomenon of antagonist-induced desensitization, and raises important issues regarding the approach to establish optimal treatment regimens for antagonist drugs.