Oxytocin has long been used as an uterotonic during labor management in women and yet responses to oxytocin infusion remain variable and unpredictable among patients. The investigation of oxytocin receptor (OTR) regulation will benefit labor management, because the clinical practice of continuous iv infusion of oxytocin is not optimal. As with other G protein-coupled receptors it is likely that the OTR internalizes and/or desensitizes upon continuous agonist exposure. The mechanisms by which this might occur however are unclear. Here we explore OTR internalization and desensitization in HEK cells by utilizing inhibitors of heterologous second messenger systems and recently available mutant cDNA constructs. We report rapid and extensive internalization and desensitization of the OTR upon agonist exposure. Internalization was unaffected by inhibitors of protein kinase C or Ca²⁺ calmodulin-dependant kinase II but was significantly reduced after transfection with dominant negative mutant cDNAs of G protein-coupled receptor kinase 2, -Arrestin2, Dynamin and Eps15 (a component of clathrin-coated pits). Moreover, desensitization of the OTR, measured by a calcium mobilization assay, was also inhibited by the aforementioned cDNA constructs. Thus, our data demonstrate for the first time the importance of the classical clathrin mediated pathway during agonist induced OTR internalization and desensitization.