The recent discovery that an RFamide termed gonadotropin-inhibitory hormone is likely to be a hypophysiotrophic gonadotropin release-inhibiting hormone in birds has generated interest into the role of LPXRFamide neuropeptides in the control of gonadotropin secretion in mammals. Recent immunocytochemical studies in birds and mammals have suggested that neurons expressing the mammalian LPXRFamides, RFamide-related peptides (RFRPs) 1 and 3, may innervate and regulate GnRH neurons directly. We used cell-attached electrophysiology in adult male and female GnRH-green fluorescent protein-tagged neurons to examine whether RFRP-3 modulated the electrical excitability of GnRH neurons. RFRP-3 was found to exhibit rapid and repeatable inhibitory effects on the firing rate of 41% of GnRH neurons. A small population of GnRH neurons (12%) increased their firing rate in response to RFRP-3, and the remainder was unaffected. No difference was detected in the RFRP-3 responses of GnRH neurons from male, diestrous, or proestrus female mice. The suppressive effect of RFRP-3 was maintained when amino acid transmission was blocked, suggesting a possible direct effect of RFRP-3 upon GnRH neurons. To evaluate the effects of other RFamide neuropeptides on GnRH neurons, we tested the actions of prolactin-releasing peptide-20 and -31. Neither compounds altered the firing rate of GnRH neurons. These studies demonstrate that RFRP-3 has a likely direct suppressive action on the excitability of GnRH neurons, indicating a role for RFRPs in the regulation of gonadotropin secretion in mammals through modulation of GnRH neuron activity.