We developed PET ligands (+)N-[(11)C]ethyl-3-piperidyl benzilate ([(11)C](+)3-EPB) and (+)N-[(11)C]propyl-3-piperidyl benzilate ([(11)C](+)3-PPB) for cerebral muscarinic cholinergic receptors. The distribution and kinetics of the novel ligands were evaluated for comparison with the previously reported ligand (+)N-[(11)C]methyl-3-piperidyl benzilate ([(11)C](+)3-MPB) in the monkey brain (Macaca mulatta) in the conscious state using high-resolution positron emission tomography (PET). At 60-91 min postinjection, regional distribution patterns of these three ligands were almost identical, and were consistent with the muscarinic receptor density in the brain as previously reported in vitro. However, the time-activity curves of [(11)C](+)3-EPB and [(11)C](+)3-PPB showed earlier peak times of radioactivity and a faster clearance rate than [(11)C](+)3-MPB in cortical regions rich in the receptors. Kinetic analysis using the three-compartment model with time-activity curves of radioactivity in metabolite-corrected arterial plasma as input functions revealed that labeling with longer [(11)C]alkyl chain length induced lower binding potential (BP = k(3)/k(4)), consistent with the rank order of affinity of these ligands obtained by an in vitro assay using rat brain slices and [(3)H]QNB. The cholinesterase inhibitor Aricept administered at doses of 50 and 250 microg/kg increased acetylcholine level in extracellular fluid of the frontal cortex and the binding of [(11)C](+)3-PPB with the lowest affinity to the receptors was displaced by the endogenous acetylcholine induced by cholinesterase inhibition, while [(11)C](+)3-MPB with the highest affinity was not significantly affected. Taken together, these observations indicate that the increase in [(11)C]alkyl chain length could alter the kinetic properties of conventional receptor ligands for PET by reducing the affinity to receptors, which might make it possible to assess the interaction between endogenous neurotransmitters and ligand-receptor binding in vivo as measured by PET. Copyright 2001 Wiley-Liss, Inc.