Photodynamic therapy (PDT) is a light-based cancer treatment modality. Here we employed both in vivo and ex vivo fluorescence imaging to visualize vascular response and tumor cell survival after verteporfin-mediated PDT designed to target tumor vasculature. EGFP-MatLyLu prostate tumor cells, transduced with EGFP using lentivirus vectors, were implanted in athymic nude mice. Immediately after PDT with different doses of verteporfin, tumor-bearing animals were injected with a fluorochrome-labeled albumin. The extravasation of fluorescent albumin along with tumor EGFP fluorescence was monitored noninvasively with a whole-body fluorescence imaging system. Ex vivo fluorescence microscopy was performed on frozen sections of tumor tissues taken at different times after treatment. Both in vivo and ex vivo imaging demonstrated that vascular-targeting PDT with verteporfin significantly increased the extravasation of fluorochrome-labeled albumin in the tumor tissue, especially in the tumor periphery. Although PDT induced substantial vascular shutdown in interior blood vessels, some peripheral tumor vessels were able to maintain perfusion function up to 24 hr after treatment. As a result, viable tumor cells were typically detected in the tumor periphery in spite of extensive tumor cell death. Our results demonstrate that vascular-targeting PDT with verteporfin causes a dose- and time-dependent increase in vascular permeability and decrease in blood perfusion. However, compared to the interior blood vessels, peripheral tumor blood vessels were found less sensitive to PDT-induced vascular shutdown, which was associated with subsequent tumor recurrence in the tumor periphery. © 2008 Wiley-Liss, Inc.