The RF-amide family of peptides has been shown to regulate various physiological functions, including food intake and reproduction. In avian species, a RF-amide peptide with suppressive effects on the release of LH has been termed gonadotropin-inhibiting hormone (GnIH) and has been proposed to function as a major regulator of reproductive seasonality. In some mammalian species, GnIH and its mammalian homologue (RF-amide-related peptide; RFRP) have been shown to suppress the secretion of LH and adenohypophyseal responsiveness to GnRH. This has led to speculation of a role for RFRP in regulating seasonal-breeding in the horse. The sequence of the equine RFRP cDNA and analysis of predicted cutting sites for the equine prepro-RFRP indicate that 3 RF-amide-related peptides can be produced, with RFRP-3 as the homologue for the avian GnIH. Three experiments were conducted to test the hypothesis that eRFRP3 negatively regulates the secretion of LH in mares. Equine RFRP-3 (IPNLPQRF-G; MW 983) was synthesized (Auspep Pty Ltd, Tullamarine Victoria, AU) and used in all experiments. In Exp. 1, Alzet osmotic pumps delivering gonadotropin-releasing hormone (GnRH) continuously at a rate of 20 µg/h were placed s.c. in 6 mares 2 d after ovulation. Mares received bolus I.V. injections of 0, 500 and 1,000 µg eRFRP3 in a replicated Latin square design during Days 4, 6 and 8 post-ovulation. Jugular blood samples were collected before and at 15-min intervals for 4 h following treatment for assay of LH. For Exp. 2, the intercavernous sinus (ICS) of follicular phase mares was catheterized via the external facial vein for sampling of pituitary venous effluent and characterization of episodic LH release. Blood samples were collected at 5-min intervals for 6 h. Mares (n = 6 per treatment) received either saline or eRFRP3 (250 µg) I.V. every 10 min for 6 h beginning 2 h after onset of sampling. At hour 6, each mare was challenged with 1000 µg GnRH and blood sampling continued at 15-min intervals for the remaining 2 h of treatment. In Exp. 3, 3 winter anovulatory mares treated continuously with GnRH (100 µg/h), which elevated circulating LH to values similar to the breeding season, were used. On Day 5, the ICS was catheterized as in Exp. 2 and mares received saline or RFRP-3 (5 mg). ICS blood samples were collected at 5-min intervals for 3 h, with a 24-h washout period between treatments. In Exp. 1, mean concentrations (ng/ml) of LH in the peripheral circulation averaged 1.2 ± 0.2 ng/ml and did not differ between groups before or following RFRP-3 treatment. In Exp. 2, 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), and duration (36.3 ± 3.5 min) of individual secretory episodes differed between groups before or after eRFRP-3 treatment. Areas under the GnRH-induced LH curve (arbitrary units) also did not differ between control and treated mares (227.4 ± 42.9 vs. 260.2 ± 36.2). Finally, a single I.V. injection of 5 mg RFRP-3 (Exp. 3) failed to alter any variable of LH release. In contrast to observations in birds and other mammals, results of the current experiments fail to provide evidence for functional activity of eRFRP-3 in regulating LH release in the mare. Supported by Texas H-9137 and the Link Equine Research Fund. (poster)