E2020 is a piperidine cholinesterase inhibitor (ChEI) which is structurally distinct from other compounds presently under study for treatment of Alzheimer's disease. We studied the effect of this compound on acetylcholine (ACh), norepinephrine (NE), dopamine (DA) and serotonin (5-HT; 5-hydroxytryptamine) by means of transcortical microdialysis in the cortex of awake rats with no ChEI in the probe. We also compared the inhibition of brain cholinesterase (ChE) by two different approaches. Following 0.5 and 2.0 mg/kg s.c. administration, the increase in ACh was 200% (30 min) and 2100% (1 hr), respectively. The maximal ChE inhibition at 30 min was 35.5% (2.0 mg/kg) and 15.6% (0.5 mg/kg) when measured as ACh hydrolysis in the diluted homogenate. After the 2.0 mg/kg dose, phosphorylation by DFP was completely blocked at 30 min. After 0.5 mg/kg, ChE phosphorylation was maximally inhibited at 30 min (56%) and declined thereafter to negligible levels by 3 hr. In addition, E2020 increased extracellular levels of catecholamines in cortex in agreement with our previous findings with carbamate ChEI. Following 2.0 mg/kg, both NE (100%) and DA (80%) were elevated, whereas after 0.5 mg/kg only NE (50%) was affected. Neither dose affected extracellular 5-HT. Thus, E2020, which inhibits brain ChE by a novel, reversible mechanism, elevates extracellular ACh in a comparable manner to other centrally-active ChEI, and this elevation of ACh is associated with stimulation of catecholamine release.