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The pivotal role of vascular endothelial growth factor (VEGF) in cancer is underscored by the approval of bevacizumab (Bev, a humanized anti-VEGF monoclonal antibody) for first line treatment of cancer patients. The aim of this study was to develop a dual-labeled Bev for both positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging of VEGF. Bev was conjugated to a NIRF dye (ie 800CW) and 2-S-(4-isothiocyanatobenzyl)-1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA) before 64 Cu-labeling. Flow cytometry analysis of U87MG human glioblastoma cells revealed no difference in VEGF binding affinity/specificity between Bev and NOTA-Bev-800CW. 64 Cu-labeling of NOTA-Bev-800CW was achieved with high yield. Serial PET imaging of U87MG tumor-bearing female nude mice revealed that tumor uptake of 64 Cu-NOTA-Bev-800CW was 4.6±0.7, 16.3±1.6, 18.1±1.4 and …