Central nociceptin/orphanin FQ (N/OFQ)-expressing neurones are abundantly expressed in the hypothalamus and limbic system and are implicated in the regulation of activity of the hypothalamic-pituitary-adrenal axis (HPA) and stress responses. We investigated the role of the endogenous N/OFQ receptor (NOP) system using the nonpeptidic NOP antagonist, JTC-801 [N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxy-methyl)benzamide monohydrochloride], during the HPA axis response to acute physical/psychological stress (60 min of restraint). Although i.v. JTC-801 (0.05 mg/kg in 100 μl) had no significant effect on restraint-induced plasma corticosterone release at 30 or 60 min post-injection, i.v. JTC-801 (0.05 mg/kg in 100 μl) in quiescent rats significantly increased basal plasma corticosterone at the 30-min time-point compared to i.v. vehicle (1% dimethysulphoxide in sterile saline). Central injection of JTC-801 i.c.v. was associated with increased Fos expression in the parvocellular paraventricular nucleus 90 min after infusion compared to vehicle control. These findings contrast to the effects of i.c.v. UFP-101, a NOP antagonist that we have previously shown to have no effect on HPA activity in quiescent rats. To determine whether restraint stress was associated with compensatory changes in N/OFQ precursor (ppN/OFQ) or NOP receptor mRNAs, in a separate study, we undertook reverse transcriptase-polymerase chain reaction and in situ hybridisation analysis of ppN/OFQ and NOP transcripts in the brains of male Sprague-Dawley rats. In support of an endogenous role for central N/OFQ in psychological stress, we found that acute restraint significantly decreased preproN/OFQ transcript expression in the hippocampus 2 h after stress compared to unstressed controls. PpN/OFQ mRNA was also reduced in the mediodorsal forebrain 4 h after stress. NOP mRNA was reduced in the hypothalamus 2 h after restraint and at 4 h in mediodorsal forebrain and hippocampus. In situ hybridisation analysis showed that acute restraint significantly decreased ppNN/OFQ in the central amygdala, with significantly increased expression in bed nucleus and reticular thalamus associated with repeated restraint. There was a strong trend for reduced NOP mRNA in the bed nucleus of acute and repeated restraint groups, although there were no other significant changes seen. Although the exact mechanisms require elucidation, the findings obtained in the present study provide evidence indicating that the endogenous N/OFQ system is involved in both acute and chronic restraint stress responses. In summary, our findings confirm the significant role of endogenous NOP receptors and tonic N/OFQ function in the response to the psychological stress of restraint.