Glioblastoma multiforme (GBM) is known to be a hypoxic tumor and hypoxia adversely affects response to radiation therapy. Dodecafluoropentane emulsion (DDFPe) on a weight basis, delivers over 100x as much oxygen as other higher molecular weight fluorocarbons and is under study as an oxygen therapeutic in GBM patients treated with chemoirradiation. Tissue oxygen level dependent (TOLD) MRI is an oxygen sensitive imaging technique that is being used to study tumor re-oxygenation in these patients.With informed consent and IRB approval adult patients with GBM with residual tumor post-surgery were enrolled into the dose escalation phase of the study. Patients received DDFPe (2%w/vol DDFPe) at doses of 0.05 cc/kg, 0.1 cc/kg or 0.17 cc/kg I.V. 30-60 minutes prior to each fraction of radiation (2-Gy each, 30 fractions over 6 weeks). Temozolomide chemotherapy was given concurrently. Radiation was performed while the patients were breathing carbogen via a non-rebreathing circuit; the post-radiation MRI was performed while the patients were breathing oxygen. All patients underwent standard MR imaging at 1.5 Tesla. TOLD MRI was performed on patients at the higher two dose levels pre and post DDFPe on two separate occasions, days #1 and days #5 or 10 after initiation of radiation. Steady state free precession (SSFP) Look-Locker was used to obtain T₁ maps. T₁ maps were calculated from 3 axial slices obtained through tumor from either 3 or 4 repetitions. TOLD MRI showed substantial decreases in T₁ of tumor tissue after dosing with DDFPe and breathing oxygen (mean T₁ decreased from 1501±454 ms to 1371±35 ms on day one and 1459±326 ms to 1183±271 ms on day 5 for the patient receiving 0.1 cc/kg DDFPe; from 1511±93 ms to 1250±85 ms on day 1 and from 1454±75 ms to 1281±77 ms on day 10 for the patient receiving 0.17cc/kg DDFPe), while inconsistent changes were found in contralateral normal brain (ranging from 863 to 1144 ms for both patients at all time points). The two different doses of DDFPe showed similar effect. The change in T₁ (TOLD) with oxygen breathing after DDFPe administration indicated improved tumor oxygenation. The utility of TOLD MRI as a potential non-invasive prognostic imaging biomarker holds promise for precision medicine in terms of assessing tissue reoxygenation of hypoxic tumors. The ability to alter tumor oxygenation in GBM patients pre-treated with DDFPe could have important therapeutic implications.