Oviducts play roles in reproductive processes, including gametes transport, fertilization and early embryo development. Oviductal transport is controlled by various factors such as endothelins (EDNs) and nitric oxide (NO), smooth muscle contracting and relaxing factor, respectively. EDNs and NO production depend on an ovarian steroid hormone, oestradiol-17β (E2) and E2 quickly exerts their biological functions through G protein-coupled oestrogen receptor 1 (GPER1), which mediates rapid intracellular signalling. Because follicular fluid which contains a high concentration of E2 enters the oviduct, we hypothesized that E2 in the follicular fluid participates via GPER1 in producing EDNs and NO. To test this hypothesis, we investigated 1) the expression and localization of GPER1 in bovine oviductal tissues and 2) rapid effects of E2 via GPER1 on EDN1, EDN2 and inducible NO synthase (iNOS) expression in cultured bovine oviductal isthmic epithelial cells. GPER1 was observed in the oviductal epithelium, stroma and smooth muscle, and its expression was highest in the isthmus. Short-term treatments (≤1 hr) of E2 increased EDN2 mRNA expression in the isthmic epithelial cells, although E2 did not affect EDN1 and iNOS mRNA expressions. Results of GPER1-selective agonist G-1 and GPER1-selective antagonist G-15 treatments revealed acute stimulation by E2, which is mediated via GPER1. The overall findings suggested that E2 in follicular fluid rapidly stimulates EDN2 expression via GPER1 in the isthmic epithelial cells. Follicular fluid may play a role in retention of the ovulated oocyte in the end of ampulla by contracting the isthmus for successful fertilization.