In photodynamic therapy (PDT), treatment efficacy may be reduced by the presence of pre-existing tumor hypoxia or by oxygen depletion during the therapy. Tumor oxygenation during PDT has been measured with needle electrodes, but the intratumoral distribution of this oxygen is not known. In the present study, the spatial distribution of hypoxia during PDT was quantified using the hypoxia-labeling marker EF3. Mice bearing radiation-induced fibrosarcoma tumors were treated with Photofrin-mediated PDT to a total dose of 135 J/cm(2), delivered at a fluence rate of either 75 mW/cm(2) or 38 mW/cm(2). PDT-created hypoxia at each fluence rate was labeled by exposing tumors to EF3 (52 mg/kg) during the period of illumination. Cryosectioning, immunohistochemistry, and fluorescence microscopy were carried out to quantify EF3 binding as a function of distance to the nearest perfused blood vessels in sections cut from within the superficial (light-adjacent) 600 micro m or the deep (light-distant) 600 micro m of tumors (5-6 mm in diameter, approximately 3 mm in depth). In both superficial and deep sections, PDT at 75 mW/cm(2) resulted in the development of significant gradients in tumor hypoxia as a function of distance to a perfused blood vessel. Furthermore, significant hypoxia was detected even in vascular-adjacent tissue. These effects were associated with a significant decrease in the percentage of perfused vessels and a significant increase in the median distance of a cell to the nearest perfused blood vessel. In contrast, during PDT at 38 mW/cm(2), sections from deep tumor levels demonstrated only insignificant increases in the rise in hypoxia as a function of distance to a perfused vessel and in the level of hypoxia in vascular-adjacent tissue. No effects on tumor perfusion were detected during PDT at 38 mW/cm(2). Overall, these results demonstrate that spatially dependent depletion of oxygen can occur during PDT as a function of the fluence rate and that PDT can create significant hypoxia in even tissue adjacent to perfused blood vessels.