Photodynamic therapy (PDT) requires oxygen to cause tumor damage, yet therapy itself can deplete or enhance tumor oxygenation. In the present work we measured the PDT-induced change in tumor oxygenation and explored its utility for predicting long-term response to treatment. The tissue hemoglobin oxygen saturation (SO(2)) of murine tumors was noninvasively measured by broadband diffuse reflectance spectroscopy. In initial validation studies, the oxyhemoglobin dissociation curve for mouse blood was accurately recreated based on measurements during deoxygenation of a tissue phantom of mouse erythrocytes. In vivo studies exhibited excellent correlation between carbogen-induced changes in SO(2) and pO(2) of radiation-induced fibrosarcoma tumors measured by reflectance spectroscopy and the Eppendorf pO(2) histograph, respectively. In PDT studies radiation-induced fibrosarcoma tumor SO(2) was measured immediately before and after Photofrin-PDT (135 J/cm(2), 38 mW/cm(2)). Animals were subsequently followed for tumor growth to a volume of 400 mm(3) (time-to-400 mm(3)) or the presence of tumor cure (no tumor growth at 90 days after treatment). In animals that recurred, the PDT-induced change in tumor SO(2), i.e., relative-SO(2) (SO(2) after PDT/SO(2) before PDT) was positively correlated with treatment durability (time-to-400 mm(3)). The predictive value of relative-SO(2) was confirmed in a second group of animals with enhanced pre-PDT oxygenation due to carbogen breathing. Furthermore, when all of the animals were considered (those that recurred and those that were cured) a highly significant association was found between increasing relative-SO(2) and increasing probability of survival, i.e., absence of recurrence. As independent variables, the SO(2) after PDT, the pre-PDT tumor volume, and light penetration depth all failed to predict response. As an independent variable, the SO(2) before PDT demonstrated a weak negative association with treatment durability; this association was driven by a correlation between decreasing pre-PDT SO(2) and increasing relative-SO(2). These data suggest that monitoring of PDT-induced changes in tumor oxygenation may be a valuable prognostic indicator.