Orphanin FQ is a novel opioid family member, which is densely localized in the hypothalamus, a region of the brain important for the control of reproduction. This study tested the hypothesis that orphanin FQ might regulate the secretion of gonadotropin-releasing hormone, the key central regulator of the ovulatory cycle. To test this hypothesis, we used rat hypothalamic fragments and immortalized gonadotropin-releasing hormone neurons (GT1-7) in vitro and examined whether orphanin FQ would inhibit forskolin-induced gonadotropin-releasing hormone release. The studies revealed that orphanin FQ potently and dose-dependently inhibits forskolin-induced gonadotropin-releasing hormone release from rat hypothalamic fragments. In contrast, orphanin FQ had no effect on gonadotropin-releasing hormone release from GT1-7 neurons. Reverse transcriptase-polymerase chain reaction analysis further revealed that the orphanin FQ receptor, ORL-1 is expressed in hypothalamic fragments, but not in GT1-7 neurons. Together, these findings are the first to suggest a role for orphanin FQ in the regulation of gonadotropin releasing hormone secretion.