GnRH provides the primary stimulus for the reproductive axis, but original work also revealed the existence of a gonadotropin-inhibitory hormone (GnIH) in birds. In mammals, GnIH properties are displayed by a hypothalamic dodecapeptide, which is a member of the RF-amide family, namely RF-amide-related peptide (RFRP)-3. This peptide inhibits GnRH-stimulated gonadotropin secretion from ovine pituitary cells in culture, but it is not known whether there are effects on gonadotropin synthesis. The aim of the present study was to determine the effects of RFRP-3 on the expression of genes for beta-subunits of the gonadotropins in ovine pituitary cells from gonadectomized ewes and rams. Cells in primary culture were given GnRH or vehicle pulses every 8 h for 24 h with and without RFRP-3 treatment. GnRH stimulated LH and FSH secretion, which was reduced by RFRP-3. Quantitative real-time PCR revealed increased expression of LHbeta and FSHbeta subunit genes after GnRH treatment and a specific reduction in expression after RFRP-3 treatment. There was no effect on the expression of GH, proopiomelanocortin, or prolactin genes. Western blotting showed that GnRH stimulated phosphorylation of ERK (phospho-ERK-1/2), and this effect was abolished by RFRP-3. We conclude that RFRP-3 acts on the pituitary gonadotropes to inhibit synthesis of the gonadotropins, and this effect may be mediated by a reduction in the GnRH-stimulated second messenger phospho-ERK-1/2.