We recently labeled with carbon-11, a high affinity, selective, 5-HT3 receptor (5-HT3R) ligand, S21007, for potential positron emission tomography (PET) applications. To evaluate the in vivo binding properties of [11C]S21007, its brain regional distribution, tissue and plasma pharmacokinetics and plasma metabolisation were characterized. To circumvent the problem of highly discrete brain localization of the 5-HT3R (area postrema, hippocampus), we designed an original approach combining high-resolution imaging techniques (ex vivo phosphor plate autoradiography and MRI-guided coronal PET in the rat and baboon, respectively). After i.v. injection of trace amounts of [11C]S21007 to rats, phosphorimager autoradiography failed to reveal in vivo specific binding to, nor selectivity for 5-HT3R-rich areas. PET studies in the baboon showed consistent results, i.e., there was no selective accumulation of [11C]S21007 in the area postrema or hippocampus, and neither displacement nor presaturation with cold S21007 resulted in significant changes in tissue distribution or kinetics of [11C]S21007.