Hypericin, a polycyclic quinone obtained from plants of the genus Hypericum, has been proven to be a potent photosensitizer. The mechanism of tumor eradication and mode of cell death induced by in vivo photodynamic therapy (PDT) with hypericin were investigated in the present study using 2 therapeutic protocols. RIF-1 tumors were exposed to laser light at either 0.5 hr or 6 hr after hypericin administration (5 mg/kg, i.v.). A significant reduction in tumor perfusion, as determined by the retention of fluorescein in the tumor tissue, was detected immediately after both PDT treatments. Further decrease in tumor perfusion was observed in the hours after treatment. The re-establishment of tumor perfusion, however, occurred 24 hr after 6 hr-interval PDT, but not after 0.5 hr-interval PDT. The kinetics of tumor cell survival estimated by the in vivo/in vitro clonogenic assay revealed no or limited cell death when tumors were explanted immediately after irradiation, whereas a delayed but progressive cell death was detected when tumors remained in situ after both PDT treatments. The detection of nucleosomal DNA fragmentation by agarose gel electrophoresis or TUNEL assay and the assessment of cell morphology by light microscopy indicated that apoptosis was the most prominent tumor response to hypericin-mediated PDT. Furthermore, immunohistochemical analysis of the tumor tissue showed an increased expression of both Fas and Fas ligand after irradiation, suggesting that this cell death pathway might contribute to the overall PDT-induced apoptotic response. In conclusion, our results demonstrate that apoptosis, likely occurring as a result of vascular damage, is responsible for the tumor eradication by PDT with hypericin in this tumor model.