The pharmacological profile of the non-peptide OP4 receptor (ORL1, LC132) agonist, Ro 64-6198, was investigated, in three electrically stimulated nociceptin/orphanin FQ (NC)-sensitive preparations, namely the mouse and rat vas deferens and the guinea pig ileum. Ro 64-6198 mimicked the inhibitory effect of NC in the three preparations, while showing slow kinetics of action and a slowly reversible effect compared to the fast and immediately and completely reversible effect of the natural peptide. Ro 64-6198 displayed similar pEC50 and Emax values as NC in the mouse and rat vas deferens while it was 100-fold less potent but more efficacious (higher Emax) than NC in the guinea pig ileum. In the rat vas deferens the effects of Ro 64-6198 were antagonised by [Nphe1]NC(1-13)NH2 and J-113397 with pKB values (6.30 and 8.05, respectively) similar to those obtained against NC (6.20 and 7.77, respectively). Naloxone (1 microM) was inactive. In the guinea pig ileum a clear shift of the concentration response curve to Ro 64-6198 was obtained only using a cocktail of antagonists (naloxone + [Nphe1]NC(1-13)NH2 or naloxone + J-113397). In the mouse vas deferens the antagonists were inactive against Ro 64-6198 either when tested alone or in combination. Therefore, Ro 64-6198 behaved as a selective OP4 receptor agonist only in the rat tissue. These results suggest a physiological heterogeneity in OP4 receptors across tissues and species and may explain why, when tested in vivo, Ro 64-6198 mimics the potent anxiolytic effect of NC better in the rat than in the mouse.