Background <p>The aim of this study was to identify hypoxic regions in tumours using non-invasive imaging without specific radioligands. Tumour hypoxia, which varies during the course of radiation therapy, is an important predictor of treatment effect, therefore, frequent monitoring is required. However, positron emission tomography (PET) using hypoxia-specific radioligands is impractical to perform in every fractionated irradiation. Alternatively, blood flow can be evaluated by measuring washout rates of irradiation-induced positron-emitters in range-verification or beam-monitoring PET. Based on this, we hypothesized that the blood flow supply and kinetics (washout) of the irradiation-produced positron-emitters in the tumour could reflect the tumour oxygen status. Beam-monitoring PET for hypoxic tumour rat models was compared with conventional hypoxia PET. Glioma C6 cancer cells were transplanted into nude rats. Tumours were irradiated with a ¹²C ion beam, and beam-monitoring PET was performed using a high sensitivity total-body small-animal PET system. A compartment model was applied to the kinetic data of the irradiation produced positron-emitters. Then, ¹⁸F-labeled fluoroazomycin arabinoside (¹⁸F-FAZA) PET imaging of the hypoxia area of the tumour of the same rat was performed.</p> Results <p>The parametric images of washout rate obtained with beam-monitoring PET were in good agreement with ¹⁸F-FAZA-PET images.</p> Conclusion <p>This study demonstrated the biological washout can be used as diagnostic tool for tumour hypoxia in routine beam-monitoring PET.</p>