Background: The rationale was to develop an ablation approach to destroy regions of tumor resistant to radiation and thus reduce the time required for whole tumor ablation, while improving overall tumor control after radiotherapy. Materials and Methods: The system is composed of a micro positron emission tomography (mPET), 7T magnetic resonance imaging (MRI), and a customized MRI-compatible focused ultrasound applicator. 18F-fluoromisonidazole (18F-miso) radioactive tracer delineated hypoxic regions based on a threshold tumor/muscle activity ratio. 18F-miso PET/MRI fused images were used for targeting tumor hypoxic regions for focused ultrasound ablation. With MRI real-time temperature imaging guidance, PET-detected hypoxic regions of tumor could be selectively ablated to temperatures (T>55 oC). In vivo validation experiments were performed in SCK and 4T1 murine mammary carcinomas. In two tumor response assays, sequence dependence of combined radiotherapy and ablation was studied in the SCK tumor model. Tumor ablation was performed using a conductive probe or focused ultrasound and ionizing radiation administered in single doses of 15-20 Gy. Results: Tumor growth was abolished when ablation was applied immediately AFTER radiation while interestingly when ablation was administered immediately BEFORE radiation, there was no difference in observed growth delay compared to ablation or radiation alone. Conclusion: PET and MRI guided focused ultrasound surgery (MRgFUS) of tumor hypoxic regions is feasible and will be potentially useful for preclinical studies using ultrasound, radiation or chemotherapy. This study suggests that radiation precedes ablative therapy to avoid unwanted stress response or additional hypoxia induced by the ablation, potentially confounding the improved response potential for combined therapy.