We measured brain acetylcholinesterase activity in 16 patients with Parkinson's disease (PD), 12 patients with progressive supranuclear palsy (PSP), and 13 age-matched controls, using N-methyl-4-[11C]piperidyl acetate and positron emission tomography. Kinetic analysis was performed to calculate k3, an index of acetylcholinesterase activity. In PD patients, there was a significant reduction (-17%) of cerebral cortical k3 compared with normal controls, whereas there was only a nonsignificant reduction (-10%) of cortical k3 in PSP patients. However, there was a prominent reduction (-38%) of thalamic k3 in PSP patients compared with normal controls, whereas there was only a nonsignificant reduction (-13%) of thalamic k3 in PD patients. The results suggest that there is a loss of cholinergic innervation to the cerebral cortex in association with cholinergic innervation to the thalamus in PD, whereas there is a preferential loss of cholinergic innervation to the thalamus in PSP. When the thalamic to cerebral cortical k3 ratio was taken for each subject, PD and PSP were separated, suggesting that positron emission tomography measurement of acetylcholinesterase activity may be useful for differentiating the two similar disorders.