Binding of [18F]3-fluoro-5-[(pyridin-3-yl)ethynyl]benzonitrile ([18F]F-PEB) was evaluated in membranes and tissue sections prepared from rat, rhesus and human brain. Saturation equilibrium binding experiments with frozen brain cortex and caudate-putamen membranes of young adult rhesus and human and with cortex and striatum from rat yielded data indicative of specific high-affinity binding (KD=0.1-0.15 nM, n> or =3) to a saturable site previously shown to be metabotropic glutamate receptor 5 (mGluR5; Patel S, Ndubizu O, Hamill T, Chaudhary A, Burns HD, Hargreaves RJ, Gibson RE. Screening cascade and development of potential positron emission tomography radiotracers for mGluR5: in vitro and in vivo characterization. Mol Imaging Biol 2005;7:314-323). High-affinity binding of [18F]F-PEB was also detected in cerebellum membranes from rat, rhesus and human. The density of binding sites (Bmax) measured using [18F]F-PEB followed the rank order cortex approximately caudate-putamen/striatum>cerebellum for all three species, with the cerebellum Bmax being significantly lower than that observed in the other regions. Receptor autoradiography studies in tissue sections confirmed that the regional distribution of [18F]F-PEB in mammalian central nervous system is consistent with that of mGluR5 and that a small but specific mGluR5 signal is observed in rhesus and human cerebellum. A small and quantifiable specific signal could also be observed in rat cerebellum using this radiotracer. Immunohistochemical analysis in brain sections revealed a rank order of staining in rhesus and human brain of cortex approximately caudate-putamen>cerebellum. Rat brain immunohistochemistry followed the same rank order, although the staining in the cerebellum was significantly lower. Using a "no-wash" wipe assay, the development of a specific signal within 20 min of incubation of tissue brain sections (>60% in the cortex and striatum; 36-49% in the cerebellum) from all three species confirmed previous in vivo data from rat and rhesus monkey that [18F]PEB is likely to provide a useful in vivo signal using positron emission tomography (PET). This study provides the first quantitative demonstration and direct comparison of a PET tracer candidate identifying mGluR5 binding sites in mammalian cerebellum, which subsequently raises questions in terms of using the cerebellum as a null tissue in PET imaging studies in the laboratory and the clinic.