This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Carbetocin, a long-acting oxytocin analogue, has been reported to elicit interesting and peculiar behavioural effects. The present study investigated the molecular pharmacology of carbetocin, aiming to better understand the molecular basis of its action in the brain. Using bioluminescence resonance energy transfer biosensors, we characterised the effects of carbetocin on the three human oxytocin/vasopressin receptors expressed in the nervous system: the oxytocin receptor (OXTR) and the vasopressin V1a (V1aR) and V1b (V1bR) receptors. Our results indicate that (i) carbetocin activates the OXTR but not the V1aR and V1bR at which it may act as an antagonist; (ii) carbetocin selectively activates only the OXTR/Gq pathway displaying a strong functional selectivity; (iii) carbetocin is a partial agonist at the OXTR/Gq coupling; (iv) carbetocin promotes OXTR internalisation via a previously unreported b-arrestin-independent pathway; and (v) carbetocin does not induce OXTR recycling to the plasma membrane. Altogether, these molecular pharmacology features identify carbetocin as a substantially different analogue compared to the endogenous oxytocin and, consequently, carbetocin is not expected to mimic oxytocin in the brain. Whether these unique features of carbetocin could be exploited therapeutically remains to be established. Oxytocin is a small nonapeptide produced by magnocellular and parvocellular neurones of the supraoptic and paraventricular nuclei of the hypothalamus; from these sites of synthesis, the peptide is delivered to the periphery by axons projecting to the posterior pituitary and to the central nervous system (CNS) by dendrites and axonal collaterals (1,2). In the periphery, oxytocin has several functions, including the contraction of uterine smooth muscles during labour, an effect that has been extensively exploited for decades to promote contractions during the third stage of labour and to control bleeding after childbirth (3,4). Oxytocin also promotes the contraction of the mammary myoepithelium during lactation (5) and the intranasal use of oxytocin to stimulate lactation in breastfeeding women was approved by the Food and Drug Administration in the USA in 1960, even though it was discontinued in 1997 for commercial reasons.In the CNS, oxytocin acts as a neurotransmitter/neuromodulator regulating several aspects of social behaviour, learning and memory processes, and stress and anxiety responses (6,7). On the basis of its capability to promote social interactions in all vertebrates, including humans, oxytocin has been proposed as a clinical treatment for relieving social impairments associated with neurodevelopmental and psychiatric disorders (8,9). In the past decade, a number of clinical trials have been performed in autism and schizophrenia but, unfortunately, no consensus on the real efficacy of o...