Orphanin FQ/nociceptin (OFQ/N), the most recently identified endogenous opioid peptide, stimulates prolactin secretion in both male and female rats. OFQ/N, however, did not elicit this stimulatory effect through the mu-, delta-, or kappa-opiate receptor subtype. The role OFQ/N plays in prolactin regulation under physiological conditions and its mechanism of action are not known. The purpose of these studies was to determine the physiological significance and pharmacological specificity of the prolactin secretory response to OFQ/N. In addition, the role of the tuberoinfundibular dopaminergic (TIDA) neurons in mediating this response was examined. Opioid receptor-like-1 (ORL-1) receptors were blocked by pretreatment with compound B (Comp B), a purported OFQ/N antagonist, or receptor synthesis was disrupted by pretreatment with ORL-1 receptor antisense oligonucleotides. The prolactin secretory response to OFQ/N administration in diestrous females was measured. Furthermore, the suckling-induced prolactin response was also determined after Comp B pretreatment. TIDA neuronal activity was quantified in diestrous female rats to determine whether OFQ/N stimulates prolactin release by inhibiting TIDA neurons. OFQ/N significantly inhibited the TIDA neurons by 1 min, preceding the prolactin secretory response. Both Comp B and antisense pretreatment blocked the stimulatory effects of OFQ/N on prolactin release, and Comp B abolished the suckling-induced prolactin response. These studies indicate that OFQ/N is a potent stimulus for prolactin secretion in female rats and that it mediates this effect by rapid and transient inhibition of TIDA neuronal activity. Furthermore, OFQ/N plays a physiologically significant role in the regulation of prolactin secretion during lactation, and it mediates its effects via actions at the ORL-1 receptor subtype.