Fluorescence imaging is currently attracting much interest as a method for intraoperative tumor detection, but most current tracers lack tumor specificity. Therefore, this technique can be further improved by tumor-specific detection. With tumor-targeted antibodies bound to a radioactive label, tumor-specific SPECT or PET is feasible in the clinical setting. The aim of the present study was to apply antibody-based tumor detection to intraoperative optical imaging, using preclinical in vivo mouse models. METHODS Anti-vascular endothelial growth factor (VEGF) antibody bevacizumab and anti-human epidermal growth factor receptor (HER) 2 antibody trastuzumab were labeled with the near-infrared (NIR) fluorescence dye IRDye 800CW. Tumor uptake of the fluorescent tracers and their (89)Zr-labeled radioactive counterparts for PET was determined in human xenograft-bearing athymic mice during 1 wk after tracer injection, followed by ex vivo biodistribution and pathologic examination. Intraoperative imaging of fluorescent VEGF- or HER2-positive tumor lesions was performed in subcutaneous tumors and in intraperitoneal dissemination tumor models. RESULTS Tumor-to-background ratios, with fluorescent imaging, were 1.93 ± 0.40 for bevacizumab and 2.92 ± 0.29 for trastuzumab on day 6 after tracer injection. Real-time intraoperative imaging detected tumor lesions at even the submillimeter level in intraperitoneal dissemination tumor models. These results were supported by standard histology, immunohistochemistry, and fluorescence microscopy analyses. CONCLUSIONS NIR fluorescence-labeled antibodies targeting VEGF or HER2 can be used for highly specific and sensitive detection of tumor lesions in vivo. These preclinical findings encourage future clinical studies with NIR fluorescence-labeled tumor-specific antibodies for intraoperative-guided surgery in cancer patients.