Sprague-Dawley rats were trained to discriminate a subcutaneous injection of physostigmine (0.2 mg/kg) from a similar injection of saline in a two-lever, food-reinforced behavior paradigm. The training dose of physostigmine reduced the response rate to about 50% of that in saline sessions. The discriminative stimulus (DS) effect of physostigmine is mediated by a central cholinergic mechanism since it was antagonized by scopolamine (0.1 mg/kg), but was unaffected by methylscopolamine (1 mg/kg) or pirenzepine (3 mg/kg). Neostigmine produced predominantly saline-appropriate lever choice. Compounds which produced averages of greater than 80% responses on the physostigmine lever are: compound BM-5 (N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)-acetamide), tetrahydroaminoacridine (THA), RS-86 (2-ethyl-8-methyl-2,8-diazaspiro-(4,5)-decan-1,3-dion hydrobromide), cis-AF30 (2-methyl-spiro-(1,3-dioxolane-4,3′)-quinuclidine), and pilocarpine. In comparison, oxotremorine, aceclidine (3-acetoxyquinuclidine), arecoline, and nicotine produced a maximum average responding of 40–70% on the physostigmine lever. The DS effect of physostigmine in rats appeared to involve a greater participation of M 1 than M 2 muscarinic or the nicotinic receptor in the brain.