Human NmU-25 (hNmU-25) and other NmU analogs have been identified as ligands for two human orphan G-protein coupled receptors named hNmU-R1 and hNmU-R2. This research characterises signalling mediated by hNmU-R1 and hNmU-R2 expressed as recombinant proteins in HEK293 cells, particularly to define their G-protein coupling and the activation and regulation of signal transduction pathways. These studies show that these receptors couple to both Galphaq/11 and Galphai. Activation of either receptor type causes a PTX-insensitive activation of both phospholipase C and mitogen activated-protein kinase and a PTX-sensitive inhibition of adenylyl cyclase with sub-nanomolar potency for each. Activation of phospholipase C is sustained but despite this capacity for prolonged receptor activation, repetitive application of hNmU-25 does not cause repetitive intracellular Ca2+ signalling. Using several strategies this was shown to be a consequence of essentially irreversible binding of NmU to its receptors and that this is followed by ligand internalization. There were no apparent differences in the activation, coupling or regulation of the two hNmU-receptors. Dual coupling and irreversible binding was explored further by using NmU receptors endogenously expressed in cultured rat colonic and fundus smooth-muscle cells. Here, receptors were also shown to couple to Galphaq/11 and Galphai and the binding of NmU was also shown to be irreversible. This study also demonstrates NmU-mediated contractions of the rat colon in organ bath studies. However, unlike single-cell experiments, repetitive application of NmU did cause repetitive contractions of the rat distal colon. The reasons for this discrepancy are unexplained.