Increased uterine stretch appears to increase the risk of preterm labour, but the mechanism is unknown. The aim of this study was to identify factors that mediate the effect of stretch on human myometrium.Myometrial explants, prepared from biopsies obtained at elective caesarean delivery, were either studied acutely, or were maintained in prolonged culture (up to 65 h) under tension with either a 0.6 g or a 2.4 g mass, and compared using in vitro contractility, whole genome array, and qRT-PCR. Tissue held at tonic stretch with the 2.4 g mass for either 24 or 65 h showed increased potassium chloride (KCl)-induced and oxytocin-induced contractility compared with that held with the 0.6 g mass. Gene array identified 62 differentially expressed transcripts after 65 h exposure to increased stretch. Two probes for gastrin-releasing peptide (GRP), a known stimulatory agonist of smooth muscle, were among the top five up-regulated by stretch (3.4-fold and 2.0-fold). Up-regulation of GRP mRNA by stretch was confirmed in a separate series of 10 samples using quantitative RT-PCR (qRT-PCR) (2.8-fold, P =0.01). GRP stimulated contractions acutely when added to freshly obtained myometrial strips in 2 out of 9 cases, but Western blot demonstrated expression of the GRP receptor in 9 out of a further 9 cases. Prolonged incubation of stretched explants in the GRP antagonists PD-176252 or RC-3095 (65 and 24 h, respectively) significantly reduced KCl- and oxytocin-induced contractility.Tonic stretch of human myometrium increases contractility and stimulates the expression of a known smooth muscle stimulatory agonist, GRP. Incubation of myometrium with GRP receptor antagonists attenuates the effect of stretch. GRP may be a target for novel therapies to reduce the risk of preterm birth in multiple pregnancy.