The novel spiroimidazoline, S18616, a potent and efficacious agonist at alpha2-adrenoceptors (ARs), shows>100-fold selectivity versus alpha1-ARs, imidazoline receptors and all other sites examined. Herein, we characterized its discriminative stimulus (DS) properties in rats trained to recognise S18616 (0.01 mg/kg, s.c.) from saline. S18616 dose-dependently (0.0063-0.01) and "fully" (>or=80% "S18616" lever selection) substituted for itself. Full substitution was also acquired for the agonist, UK14,304 (0.04-0.16), while the partial agonist, clonidine (0.01-0.08), yielded sub-maximal substitution (67%). Guanfacine (0.16-1.25) and guanabenz (0.00063-0.04), preferential agonists at alpha2A-ARs, revealed full substitution for S18616. In contrast, the alpha1-AR agonists, cirazoline and ST587 (both 0.04-0.63), did not substitute. The alpha2-AR antagonists, RX821,002, atipamezole (both 0.0025-0.04) and idazoxan (0.04-0.63) blocked the S18616 DS, whereas the alpha1-AR antagonists, prazosin (0.16-0.63) and WB4101 (0.04-0.63), were inactive. Prazosin is also a preferential antagonist at alpha2B/2C- versus alpha2A-ARs and a further preferential alpha2B/2C-AR antagonist, BRL41,992 (0.63-2.5), was likewise ineffective. In contrast, the alpha2A-AR antagonist, BRL44,408 (0.04-0.16), dose-dependently abolished the S18616 DS. Finally, the "atypical" antidepressants, mirtazapine (0.16-10.0) and mianserin (0.63-10.0), which behave as antagonists at alpha2A-ARs, dose-dependently blocked the S18616 DS. In conclusion, S18616 elicits a robust DS in rats that principally reflects engagement of alpha2A-ARs. This novel procedure should prove useful in the characterisation of psychoactive drugs which interact with alpha2-ARs.