Ovine pituitary cell culture and perfusion bioassays were used to investigate gonadotrophin surge-attenuating factor (GnSAF) bioactivity in vitro. GnSAF is present in steroid-free human follicular fluid (HFF) from superovulated women and specifically attenuates gonadotrophin releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion. HFF produced dose-dependent suppression of GnRH-induced LH and basal FSH secretion, the former reflecting GnSAF bioactivity, the latter inhibin bioactivity. Heparin Sepharose chromatography of HFF removed inhibin bioactivity, but not GnSAF bioactivity, indicating that these are distinct entities. Ultrafiltration of HFF suggests that GnSAF has a bioactive form of 1030 kDa. Ovine pituitary perfusion demonstrated that GnSAF bioactivity in HFF is matched in the serum of superovulated and pregnant women. In addition, HFF and serum suppress both the initial and reserve pools of GnRHinduced LH secretion and greatly reduce the self-priming effect of GnRH in ovine pituitary tissue perfusion. In-vitro and in-vivo GnSAF bioactivity is consistent with a role for GnSAF in the gonadal feedback regulation of pituitary response to GnRH.