Phosphatidylserine (PS) is normally intracellular but becomes exposed on the luminal surface of vascular endothelial cells in tumors. It also becomes exposed on tumors cells responding to therapy. In the present study, we optically imaged exposed PS in vivo using PGN635, a novel monoclonal antibody that binds PS. The F(ab')(2) fragment of PGN635 was labeled with the near-infrared (NIR) dye, IRDye800CW. In vivo dynamic NIR imaging was performed after injection of 800CW-PGN635 into mice bearing radiation-treated or untreated U87 glioma xenografts growing subcutaneously or orthotopically. NIR optical imaging revealed a clear tumor contrast in nonirradiated subcutaneous U87 gliomas after injection of 800CW-PGN635. The tumor contrast was visible as early as 4 hours later and was maximal 24 hours later (tumor-to-normal tissue ratio [TNR] = 2.8 ± 0.7). Irradiation enhanced the tumor contrast at 24 hours (TNR = 4.0 ± 0.3). Similar results were observed for orthotopic gliomas. Localization of 800CW-PGN635 to tumors was antigen specific because 800CW-Aurexis, a control probe of irrelevant specificity, did not localize to the tumors, and preadministration of unlabeled PGN635 blocked the uptake of 800CW-PGN635. Fluorescence microscopy confirmed that 800CW-PGN635 was binding to PS-positive vascular endothelial cells in nonirradiated gliomas. Irradiation of the gliomas increased PS exposure on both tumor vascular endothelial cells and tumor cells and gave rise to an increase in tumor contrast with 800CW-PGN635 that was predictive of the reduction in tumor growth. 800CW-PGN635 may be a useful new imaging probe for detection of exposed PS in tumors responding to therapy.