In rat dorsal raphe neurones, nociceptin (300 nM) reduced the peak [Ca(2+)](i) transient, triggered by depolarization, by 36.7+/-1.8% (n=46). This effect of nociceptin decreased to 16.7+/-2.9% (n=18) after pre-treatment of the neurones with pertussis toxin (5 microg/ml, 2-6 h) but was unchanged (37.4+/-2.1%, n=44) after pre-incubation with cholera toxin (5 microg/ml, 2-6 h). This suggests that, in dorsal raphe neurones, the ORL1 receptor couples to inhibitory (G(i/o)) G-proteins. The neuropeptide FF analogue, [D-Tyr1, (N-Me)Phe(3)]neuropeptide FF (10, 100, 1000 nM), acted as an anti-opioid and reduced the effect of nociceptin (300 nM, 30 s) by 62.0+/-3.3% (n=28). Following pre-incubation with cholera toxin (5 microg/ml, 2-6 h) [D-Tyr1, (N-Me)Phe3] neuropeptide FF was unable, at the three concentrations tested, to block nociceptin activity. We conclude that, in rat dorsal raphe neurones, neuropeptide FF receptors couple to stimulatory G-proteins (Gs).