Primary cultures of ovine pituitaries from adult ewes were used to investigate aspects of gonadotrophin surge-attenuating factor (GnSAF) bioactivity in human follicular fluid (hFF) from superovulated women. During the autumn and first half of the winter, LH secretion induced by gonadotrophin-releasing hormone (GnRH) was markedly reduced (43.5 ± 5.2% of control GnRH-induced LH secretion) by incubation for 48 h with steroid-free hFF. For the rest of the year, treatment with the same batch of steroid-free hFF resulted in non-significant reduction or stimulation of GnRH-induced LH secretion (71.3 ± 13.2 to 117.8 ± 11.2% of control GnRH-induced LH secretion). Incubation of pituitary cells for 48 h with oestradiol (1 pmol/l to 1 μmol/l), progesterone (1 pmol/l to 1 μmol/l) or oestradiol and progesterone combined (1 pmol/l to 1 μmol/l) in a two-way titration for 48 h had no significant effect on GnRH-induced LH secretion (83.4 ± 7.6 to 110.6 ± 5.0% of control secretion). Separating hFF into fractions of different molecular mass by ultrafiltration demonstrated that GnSAF bioactivity was present in a form 10-30 kDa in size. Incubation for 48 h with these fractions had no significant effect on basal FSH secretion but significantly attenuated GnRH-induced LH secretion during the autumn. The same fractions had little effect on GnRH-induced LH secretion from pituitary cells collected during the summer. We conclude that ovine pituitaries display at least partial reduction in sensitivity to GnSAF outside the breeding season. In addition, neither oestradiol nor progesterone singly or in combination caused the observed attenuation of GnRH-induced LH secretion that is ascribed to GnSAF bioactivity.