The density of the Imidazoline 2 binding site (I 2BS) has been shown to change in psychiatric conditions such as depression and addiction, along with neurodegenerative disorders such as Alzheimer's disease and Huntington's chorea. The presence of I 2BS on glial cells and the possibility that they may in some way regulate glial fibrillary acidic protein has led to increased interest into the role of I 2BS and I 2BS ligands in conditions characterized by marked gliosis. In addition, it has been suggested that I 2BS may be a marker for human glioblastomas. Therefore, the development of a positron emission tomography (PET) radioligand for the I 2BS would be of major benefit in our understanding of these conditions. We now report the successful synthesis and initial pharmacological evaluation of potential PET radioligands for the I 2BS as well as the tritiation and characterization of the most favorable of the series, BU99008 (6), both in vitro and ex vivo in rat. The series as a whole demonstrated excellent affinity and selectivity for the I 2BS, with BU99008 (6) selected as the lead candidate to be taken forward for in vivo assessment. BU99008 (6) showed very good affinity for the I 2BS (K i of 1.4 nM; K d = 1.3 nM), good selectivity compared with the α 2-adrenoceptor (909-fold). In addition, following peripheral administration, [ 3H]BU99008 demonstrated a heterogenous uptake into the rat brain consistent with the known distribution of the I 2BS in vivo. This, and the amenability of BU99008 (6) to radiolabeling with a positron-emitting radioisotope, indicates its potential as a PET radioligand for imaging the I 2BS in vivo.