Impairment in blood-to-brain transport of leptin is a major cause as well as consequence of obesity. Leptin crosses the blood-brain barrier by transcytosis rather than undergoing intracellular degradation. Results from previous studies have indicated that the membrane juxtapositional cytoplasmic sequence of the leptin receptor ObR is responsible for leptin transport. To identify the specific structural domains, we generated a series of ObR truncates with different lengths of the intracellular sequence, overexpressed them in 3 types of mammalian cells including cerebral endothelia, and quantified leptin binding and endocytosis. All mutant ObRs were able to bind and mediate the internalization of leptin. Surprisingly, ObR860, a construct with no cytoplasmic sequence, could act like the classical ObRa transporter in internalizing leptin. There were some cell type-dependent variations in the intracellular trafficking of Alexa-labeled leptin when mediated by ObR860 or ObRa because of differential involvement of membrane microdomains, as shown by use of the clathrin inhibitor chlorpromazine and the dynamin inhibitor Dynasore. The clathrin- and dynamin-mediated endocytosis of leptin contrasts with the lack of effect of the caveolae inhibitors nystatin and filipin. Thus, leptin-induced internalization of the ligand-receptor complex can occur without specific sorting signals in the cytoplasmic region of ObR. This novel finding may have significant implications for leptin transport.