The effects of two consecutive intramuscular doses of three cholinesterase inhibitors (physostigmine, tetrahydroaminoacridine and metrifonate) were compared in rats. The results revealed major differences in biochemical effects on the brain of the rat including the extent and duration of inhibition of cholinesterase, inhibition of release of acetylcholine and increase in levels of acetylcholine. Side effects were also markedly different in the time of appearance, duration and severity. These results suggest that there are significant differences in the mechanisms of action of various cholinesterase inhibitors. Since all three cholinesterase inhibitors are currently used in the experimental treatment of Alzheimer's disease, these findings have potential implications for the symptomatic therapy of these patients.