[3H]Hemicholinium-3 ([3H]HC-3) binding, as a marker of the presynaptic high-affinity choline uptake carrier (HACU), and cholinergic muscarinic receptors were measured by autoradiography in several brain regions of levodopa-responsive parkinsonism and matched cases. A significant increase in the density of [3H]HC-3 binding sites was found in the striatum of parkinsonian brains, while there was a slight decrease in the parkinsonian hippocampus. Total, M1 and non-M1 muscarinic receptors remained unchanged in frontal cortex and striatum of parkinsonian brains as compared to controls. Total and non-M1 muscarinic receptors were significantly reduced in the parkinsonian hippocampus, whereas hippocampal M1 receptors were preserved. These data demonstrate a hyperactivity of the HACU, and thus of the acetylcholine synthesis, in parkinsonian brains probably compensatory of the loss of both dopaminergic terminals in the striatum and of basal forebrain neurons in the hippocampus. Our results emphasize the value of [3H]HC-3 binding in the study of the functional status of the cholinergic synapse in neurodegenerative disorders.