Acetylcholine is found within key sectors of the olfactory pathway, and is the neurotransmitter for many bulbopetal axons terminating in the glomerular and internal plexiform layers of the olfactory bulb. The present study determined whether systemically administered physostigmine, a cholinesterase inhibitor, alters the rat's ability to discern among odorant mixtures. Following appropriate training, the performance of eight rats was measured every third day on an odor mixture discrimination test with six levels of difficulty. On each test day (separated from one another by 3 days), a different drug treatment was administered [i.e., 0.00 (saline), 0.05, 0.10, or 0.20 mg/kg physostigmine]. The presentation order of the treatments was counterbalanced across subjects using 4 x 4 Latin squares. The mixture discrimination test consisted of discerning the odor of an airstream coming from the saturated head space of a 1% concentration of ethyl acetate from an airstream saturated with a 1% concentration of ethyl acetate and various concentrations of butanol (i.e., 1, 0.5, 0.1, 0.05, 0.01, or 0.005%). Physostigmine was found to enhance odor discrimination performance on the more difficult discrimination tasks in a dose-related manner, suggesting that cholinergic activation may sharpen the ability of rats to discern subtle differences among complex odor stimuli.