A sensitive and specific heterologous radioimmunoassay for FSH-suppressing protein (FSP or follistatin) was applied to ovine plasma. Following a logit-log dose transformation, parallel dose-response lines were observed between purified bovine 35 kDa FSP used as standard and serial dilutions of ewe plasma. Activin-A, inhibin-A and a range of other proteins showed low (< 0.5%) cross-reactivity in the assay. Daily variations in the peripheral concentrations of FSP were measured across the ovine oestrous cycle. The peripheral concentrations of plasma FSP in adult ewes revealed a significant (P < 0.01) increase (33%) during the luteal phase above follicular phase levels, peaking 10 days after the LH surge. FSP concentrations were determined in arterial and venous plasma from the ovary, head, kidney and liver. A significant (P < 0.05) increase across the ovary was detected with no significant differences across the head, liver and kidney. To investigate the relationship between gonadal FSP and the pituitary, ewes underwent ovariectomy and hypophysectomy. FSP levels rose (100-110%, P < 0.01) during the period of surgery for both bilateral ovariectomy and sham ovariectomy, and then decreased significantly (37-44%) at 4-6 h after surgery. A further rise in plasma FSP (180-200% increase above pretreatment levels, P < 0.001) was observed 10-12 h after ovariectomy and sham ovariectomy. FSP levels then returned to preoperative levels during the following 26 h. Plasma FSP levels in long-term ovariectomized and hypophysectomized ewes were not significantly different from preoperative levels. To determine whether the pattern of plasma FSP seen during the ovariectomy study was due to the effect of the induction of anaesthesia, ewes were treated with sodium thiopentone and halothane or 0.9% (w/v) NaCl by procedures of similar duration to that used during surgery. Both treatments resulted in an elevation of FSP levels (33-62%) over pretreatment values only at the time of induction of anaesthesia. To examine further whether this rise in plasma FSP observed after anaesthesia was due to a stress response and therefore under the control of the pituitary-adrenal axis, ewes were treated with ACTH, dexamethasone or saline only. A further group of sheep were exposed to a barking dog for 10 min. No change in FSP levels compared with pretreatment levels or saline-treated controls were noted following any of these treatments. It was concluded that (1) FSP is present in the peripheral circulation; (2) ovariectomy contributes to plasma FSP, but no sicernible contribution to circulating levels was evident from the head, liver or kidney; (3) plasma FSP levels rose significantly during the luteal phase of the ovine oestrous cycle; and (4) FSP secretion may be associated with a stress response, perhaps related to animal handling and intensive blood sampling through mechanisms that are as yet unclear.