Four experiments were conducted to test the hypothesis that RF-amide related peptide 3 (RFRP3) negatively regulate the secretion of LH in mares. In Exp. 1, mares received native gonadotropin-releasing hormone (GnRH) continuously at a rate of 20 microgram/h, delivered subcutaneously using Alzet osmotic pumps during the luteal phase of the estrous cycle. Mares were treated with i.v. bolus injections of 0, 500 and 1,000 microgram eRFRP3 on days 4, 6 and 8 of cycle. Mean concentrations of LH in the peripheral circulation averaged 1.2 +/- 0.2 ng/mL and did not differ among groups before or following RFRP3 treatment. In Exp. 2, pituitary venous effluent was sampled for characterization of episodic release of LH. Mares received either saline or eRFRP3 (250 microgram) i.v. every 10 min for 6 h beginning 2 h after onset of sampling. At hour 6, each mare was challenged with 1 mg GnRH. Neither mean ICS concentrations of LH (1.3 +/- 0.2 ng/ml), nor frequency (3.6 +/- 0.55 episodes/h), amplitude (0.2 +/- 0.03 ng/ml), or duration (36.3 +/- 3.5 min) of individual secretory episodes, differed between groups before or after eRFRP3 treatment. Area under the GnRH-induced LH curve (arbitrary units) also did not differ between control and RFRP3 treated mares (175.9 +/- 11.4 vs. 192.6 +/- 10.6). In Exp. 3, winter anovulatory mares (n=6) were treated continuously for 7 d with GnRH (100 microgram/h) to stimulate synthesis of LH and increase circulating concentrations of LH to values similar to the breeding season. The ICS was catheterized for blood sampling and mares were treated with saline or RFRP3 (5 mg) in a replicated Latin square design. Treatment with RFRP3 failed to alter ICS mean concentration of LH (0.95 +/- .03 ng/ml). Finally in Exp. 4, mares in the follicular phase of the estrous cycle were assigned randomly to receive either saline (n=3) or 10 microgram/kg BW of oRFRP3 (n=3) in a single injection. No effect on mean concentration of LH was observed. In contrast to observations in birds and other mammals, results of the current experiments fail to provide evidence for functional activity of eRFRP3 or oRFRP3 in regulating LH release in the mare.