The administration of a perfluorochemical emulsion and carbogen (95% O2, 5% CO2) breathing before photodynamic therapy (PDT) was studied to determine how increased levels of tumor oxygenation may affect PDT-induced tumor destruction. C3H/HeJ mice bearing the RIF tumor were given injections of 5 to 10 mg/kg of dihematoporphyrin ethers 24 h prior to treatment. Animals were given injections of 12 ml/kg of Fluosol-DA (20%) followed by carbogen breathing or 12 ml/kg of saline and air breathing (controls) 1 h before tumors were exposed to 135 J/cm2 of 630-nm light treatment. Changes in the hypoxic fraction of tumors, the time course for decreases in tumor cell clonogenicity, and tumor response were measured immediately and at various times after treatment. The administration of Fluosol-DA (20%) and carbogen breathing was found to delay the onset of PDT-induced hypoxia through the first hour posttreatment. Progressive tumor hypoxia was observed after 4 h posttreatment. The time period in which tumors remained well oxygenated coincided with observations of increased tumor cell survival. Decreases in tumor cell clonogenicity were observed only after tumor cells became hypoxic. These findings were consistent with the 24-h delay in complete tumor response in animals given Fluosol-DA (20%) and carbogen breathing before PDT. There were only minor variations in long-term tumor response and cure observed between the two groups tested. A second series of experiments was done to assess any treatment advantage of the adjuvant use of Fluosol-DA (20%) and carbogen breathing with PDT at high tumor photosensitizer levels. At an injected dose of 50 mg/kg of dihematoporphyrin ethers, no such advantage was observed. The administration of Fluosol-DA (20%) and carbogen breathing did not reduce the extent of PDT-induced microvascular damage, maintain high levels of tumor oxygenation through light treatment, or modify the extent of tumor cell kill following treatment.