RF-amide related peptides (RFRP), as putative mammalian orthologs of the avian gonadotropin-inhibitory hormone (GnIH), have been proposed as key regulators of gonadotropin secretion in higher vertebrates. Yet considerable debate has arisen recently on their physiological relevance and potential mechanisms and sites of action. Present studies were undertaken to further characterize the effects of RFRP on LH and FSH secretion by a combination of in vivo and in vitro approaches in male and female rats. Initial screening via intracerebroventricular (icv) administration of different analogs of RFRP1 (RFRP1-12 and RFRP1-20) and RFRP3 (RFRP3-8 and RFRP3-17), as well as the related neuropeptide FF (NPFF8), to gonadectomized (GNX) female rats evidenced significant, albeit modest, inhibitory effects on LH secretion only for RFRP3-8 and RFRP3-17, which were detectable at the high dose rage (1 nmol for RFRP3-8, 5 nmol for RFRP3-17). This moderate inhibitory action was also documented after icv administration of RFRP3-8 to intact and GNX male rats. In addition, systemic (intravenous) administration of RFRP3-8 decreased the circulating levels of both gonadotropins in GNX male rats. Likewise, RFRP3-8 inhibited basal and GnRH-stimulated LH secretion by pituitaries from GNX males in vitro. This inhibitory effect was blocked by the antagonist of RFRP receptors, RF9. In summary, our results support a putative inhibitory role of RFRP3 as ortholog of GnIH in the regulation of gonadotropin secretion in mammals, which appears to involve direct pituitary actions as well as potential central (hypothalamic) effects.