As in the case of most G-protein coupled receptors (GPCRs), agonist stimulation of human oxytocin receptors (OTRs) leads to desensitisation and internalisation; however, little is known about the subsequent intracellular OTR trafficking, which is crucial for re-establishing agonist responsiveness.We examined receptor resensitisation by first using HEK293T cells stably expressing human OTRs. Upon agonist activation, the receptors were almost completely sequestered inside intracellular compartments that were not labelled by lysosomal markers, thus indicating that the internalised receptors were not sorted to these degrading organelles. Binding and fluorescence assays showed that almost 85% of the receptors had returned to the cell surface after four hours, by which time cell responsiveness to the agonist was also completely restored, as shown by measuring phospholipase C (PLC) activation. Similar results were also obtained in the presence of cycloheximide, thus indicating that receptor recycling and not de novo receptor synthesis was responsible for the resensitisation. Notably, very similar internalisation and recycling kinetics were observed in endogenous OTRs expressed on myometrial cells. Subsequent investigations of receptor recycling pathways showed that OTRs localise in vesicles containing the Rab5 and Rab4 small GTPases (markers of the "short cycle"), whereas there was no co-localisation with Rab11 (a marker of the "long cycle") or Rab7 (a marker of vesicles directed to endosomal/lysosomal compartments). Taken together, these data indicate that OTRs are capable of very efficient and complete resensitisation due to receptor recycling via the "short cycle". We also investigated the role of -arrestin2 in OTR recycling as these receptors have been previously classified as slowly- or non-recycling receptors on the basis of their stable association with the interacting protein. Our data suggest that the stable OTR/-arrestin2 interaction plays an important role in determining the rate of recycling of human OTRs, but does not determine the fate of endocytosed receptors.