The discriminative stimulus (cue) property of nicotine was studied in a T-maze paradigm, and the results were analyzed by a new statistical method. For rats trained on 0.4 mg/kg, the ED50 was 0.11 mg/kg. The enantinomer of natural nicotine (+)nicotine was much less potent, and both position isomers of nicotine were inactive. Anabasine, which is active at nicotinic cholinergic receptors, provided the nicotine cue. Cytisine, a potent nicotinic agonist in vitro, was ineffective after SC administration and this was shown to be due to its inability to enter the brain in adequate amounts. High doses of cytisine by the intracerebroventricular route partially provided the cue. The cue was blocked by low doses of mecamylamine and pempidine and by high doses of hexamethonium. The data indicate that the cue receptor is pharmacologically similar to the nicotinic cholinergic receptor in autonomic ganglia.