© 2015, World Molecular Imaging Society. Purpose: Copper-64 (Cu-64) and Galium-68 (Ga-68) radiolabeled DO3A and NODA conjugates of exendin-4 were used for preclinical imaging of pancreatic β cells via targeting of glucagon-like peptide-1 receptor (GLP-1R). Procedures: DO3A-VS- and NODA-VS-tagged Cys40exendin-4 (DO3A-VS-Cys40-exendin-4 and NODA-VS-Cys40-exendin-4, respectively) were labeled with Cu-64 and Ga-68 using standard techniques. Biodistribution and dynamic positron emission tomography (PET) were carried out in normal Sprague–Dawley (SD) rats. Ex vivo autoradiography imaging was conducted with freshly frozen pancreatic thin sections. Results: DO3A-VS- and NODA-VS-Cys40-exendin-4 analogues were labeled with Cu-64 and Ga-68 to a specific activity of 518.7 ± 3.7 Ci/mmol (19.19 ± 0.14 TBq/mmol) and radiochemical yield above 98 %. Biodistribution data demonstrated pancreatic uptake of 0.11 ± 0.02 %ID/g for [64Cu]DO3A-VS-, 0.14 ± 0.02 %ID/g for [64Cu]NODA-VS-, 0.11 ± 0.03 for [68Ga]DO3A-VS-, and 0.26 ± 0.03 for [68Ga]NODA-VS-Cys40-exendin-4. Excess exendin-4 and exendin-(9–39)-amide displaced all four Cu-64 and Ga-68 labeled exendin-4 derivatives in blocking studies. Conclusions: [64Cu]/[68Ga]DO3A-VS-Cys40- and [64Cu]/[68Ga]NODA-VS-Cys40-exendin-4 can be used as PET imaging agents specific for GLP-1R expressed on β cells. Here, we report the first evidence of pancreatic uptake visualized with exendin-4 derivative in a rat animal model via in vivo dynamic PET imaging.