R107474, 2-methyl-3-[2-(1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one, was investigated using in vitro and in vivo receptor assays and proved to be a potent and relatively selective α2-adrenoceptor antagonist. Performed assays in vitro were inhibition of binding to a large number of neurotransmitter receptor sites, drug receptor binding sites, ion channel binding sites, peptide receptor binding sites, and the monoamine transporters in membrane preparations of brain tissue or of cells expressing the cloned human receptors. The compound has subnanomolar affinity for hα2A- and hα2C-adrenoceptors (Ki = 0.13 and 0.15 nM, respectively) and showed nanomolar affinity for the hα2B-adrenoceptors and 5-hydroxytryptamine7 (h5-HT7) receptors (Ki = 1 and 5 nM, respectively). R107474 interacted weakly (Ki values ranging between 81 and 920 nM) with dopamine-hD2L, -hD3 and -hD4, h5-HT1D-, h5-HT1F-, h5-HT2A-, h5-HT2C-, and h5-HT5A receptors. The compound, tested up to 10 μM, interacted only at micromolar concentrations or not at all with any of the other receptor or transporter binding sites tested in this study. In vivo α2A- and α2C-adrenoceptor occupancy was measured by ex vivo autoradiography 1 h after subcutaneous (sc) administration of R107474. It was found that R107474 occupies the α2A- and α2C-adrenoceptors with an ED50 (95% confidence limits) of 0.014 mg/kg sc (0.009-0.019) and 0.026 mg/kg sc (0.022-0.030), respectively. Radiolabeled 2-methyl-3-[2-([1-11C]-1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one ([11C]R107474) was prepared and evaluated as a potential positron emission tomography (PET) ligand for studying central α2-adrenoceptors. [11C]R107474 was obtained via a Pictet-Spengler reaction with [11C]formaldehyde in 33 ± 4% overall decay-corrected radiochemical yield. The total synthesis time was 55 min and the specific activity was 24-28 GBq/μmol. The biodistribution of [11C]R107474 in rats revealed that the uptake of [11C]R107474 after in vivo intravenous administration is very rapid; in most tissues (including the brain) it reaches maximum concentration at 5 min after tracer injection. In agreement with the known distribution of α2-adrenoceptors in the brain, highest uptake of radioactivity was observed in septum (3.54 ± 0.52 ID/g, 5 min pi) and entorhinal cortex (1.57 ± 0.10 ID/g, 5 min pi). Tissue/cerebellum concentration ratios for septum (5.38 ± 0.45, 30 min pi) and entorhinal cortex (3.43 ± 0.24, 30 min pi) increased with time due to rapid uptake followed by a slow washout. In vivo blocking experiments using the non-selective α2-adrenoceptor antagonist mirtazapine demonstrated specific inhibition of [11C]R107474 binding in selective brain areas. The receptor binding profile of mirtazapine is reported and the selectivity of inhibition of binding is discussed. These results suggest that [11C]R107474 deserves further investigation as a potential radioligand for studying α2-adrenoceptors using PET.