Abstract Introduction The positron emission tomography (PET) tracer 9-18 Ffluoroethyl-(+)-dihydrotetrabenazine (18 F-FE-(+)-DTBZ) is a potential candidate for quantifying beta-cell mass in vivo. The purpose was to investigate in vitro and in vivo utility of this tracer for the assessment of beta-cell mass. Methods Three pigs were intravenously administered 18 F-FE-(+)-DTBZ and examined by PET/computed tomography. Binding parameters were estimated by kinetic modeling. In vitro kD and Bmax were determined by saturation binding studies of endocrine and exocrine human tissue homogenates. In vitro pancreatic uptake was determined by tissue autoradiography with pancreases from patients with types 1 (T1DM) and 2 diabetes mellitus (T2DM) and healthy controls. Results 18 F-FE-(+)-DTBZ had a kD of 3.5±1.0 nM, a Bmax of 382±108 fmol/mg protein and a specificity of 89±1.8% in islet homogenates. The total exocrine uptake was lower and 65% was nondisplaceable. No uptake difference was observed in pancreatic tissue slices from patients with T1DM, T2DM or healthy controls. The in vivo porcine pancreatic uptake reached a peak of standardized uptake value (SUV) of 2.8 with a low distribution volume ratio in all animals. Moderate to high tracer uptake was identified in the bile system and in bone. Conclusions 18 F-FE-(+)-DTBZ binds to vesicular monoamine transporter 2 (VMAT2) with high specificity in pure islet tissue in vitro. However, there is high nondisplaceable binding to exocrine tissue. In addition, in vivo tracer metabolism and dehalogenation result in severe underestimation of porcine pancreatic VMAT2 expression and BCM. The results do not support 18 F-FE-(+)-DTBZ as a suitable tracer for in vivo beta-cell imaging.