Ovine and rat pituitary bioassays for gonadotrophin surge-attenuating factor (GnSAF) were utilized to determine whether the level of GnSAF bioactivity in pooled human follicular fluid (hFF) from superovulated women varied according to follicle diameter (< or = 11 mm, 12-15 mm and 16-21 mm follicles examined using the ovine bioassay, or < or = 10 mm, 11-13 mm, 14-17 mm, 18-20 mm, 21-24 mm and > or = 25 mm follicles examined using the rat bioassay). When tested using dispersed ovine pituitary cells, GnSAF bioactivity, expressed in terms of the reduction in gonadotrophin-releasing hormone (GnRH)-induced LH secretion, was inversely related to follicle diameter (P < 0.01). In response to 5 microliters hFF/well from follicles of < or = 11, 12-15 and 16-21 mm diameter, GnRH-induced LH secretion was reduced to 40.5 +/- 6.9%, 65.2 +/- 6.6% and 83.7 +/- 7.9% of control respectively. A similar inverse relationship was observed when a second batch of hFF samples from different sized follicles was tested using rat pituitary cell monolayers. Expressing GnSAF bioactivity in terms of the dose required to suppress GnRH-induced LH secretion by rat pituitary cells to 50% of the maximal suppression observed (ED50), the three smallest follicle size pools contained the most GnSAF (ED50 values of 0.13, 2.79 and 5.36 microliters hFF/well from follicles of < or = 10, 11-13 and 14-17 mm respectively). The ED50 values for follicles of 18-20, 21-24 and > or = 25 mm were 8.81, 27.1 and 60.0 microliters hFF/well respectively. Thus hFF from follicles < or = 11 mm was over 450 times more potent than hFF from follicles > or = 25 mm in suppressing GnRH-induced LH release. The ED50 values for inhibin bioactivity (measured as the suppression of basal FSH secretion from rat pituitary monolayers) were much less variable than those for GnSAF bioactivity (between 0.85 and 0.13 microliters hFF/well). Inhibin immunoreactivity, measured by a two-site immunoradiometric assay, followed the same pattern as inhibin bioactivity with lowest concentrations in the smallest follicles (41.96 ng/ml) and highest concentrations in the three largest follicle size groups (56.48-64.48 ng/ml). The specific effects of inhibin on GnRH-induced LH and basal FSH release in these pituitary bioassays were determined by incubating culture dishes with pure recombinant human inhibin at doses of 0.025-25 ng/well. In both the sheep and rat pituitary monolayers, basal FSH was suppressed (ED50 = 0.02 and 0.16 ng/well respectively).(ABSTRACT TRUNCATED AT 400 WORDS)