Tumor hypoxia impedes the outcome of radiotherapy. As the extent of hypoxia in solid tumors varies during the course of radiotherapy, methods that can provide repeated assessment of tumor pO₂ such as EPR oximetry may enhance the efficacy of radiotherapy by scheduling irradiations when the tumors are oxygenated. The repeated measurements of tumor pO₂ may also identify responders, and thereby facilitate the design of better treatment plans for nonresponding tumors. We have investigated the temporal changes in the ectopic 9L and C6 glioma pO₂ irradiated with single radiation doses less than 10 Gy by EPR oximetry. The 9L and C6 tumors were hypoxic with pO₂ of approximately 5–9 mmHg. The pO₂ of C6 tumors increased significantly with irradiation of 4.8–9.3 Gy. However, no change in the 9L tumor pO₂ was observed. The irradiation of the oxygenated C6 tumors with a second dose of 4.8 Gy resulted in a significant delay in growth compared to hypoxic and 2 Gy × 5 treatment groups. The C6 tumors with an increase in pO₂ of greater than 50% from the baseline of irradiation with 4.8 Gy (responders) had a significant tumor growth delay compared to nonresponders. These results indicate that the ectopic 9L and C6 tumors responded differently to radiotherapy. We propose that the repeated measurement of the oxygen levels in the tumors during radiotherapy can be used to identify responders and to design tumor oxygen guided treatment plans to improve the outcome.