PURPOSE: There is a clinical need for noninvasive, nonionizing imaging biomarkers of tumor hypoxia and oxygenation. We evaluated the relationship of T1 -weighted oxygen-enhanced magnetic resonance imaging (OE-MRI) measurements to histopathology measurements of tumor hypoxia in a murine glioma xenograft and demonstrated technique translation in human glioblastoma multiforme. METHODS: Preclinical evaluation was performed in a subcutaneous murine human glioma xenograft (U87MG). Animals underwent OE-MRI followed by dynamic contrast-enhanced MRI (DCE-MRI) and histological measurement including reduced pimonidazole adducts and CD31 staining. Area under the curve (AUC) was measured for the R1 curve for OE-MRI and the gadolinium concentration curve for DCE-MRI. Clinical evaluation in five patients used analogous imaging protocols and analyses. RESULTS: Changes in AUC of OE-MRI (AUCOE ) signal were regionally heterogeneous across all U87MG tumors. Tumor regions with negative AUCOE typically had low DCE-MRI perfusion, had positive correlation with hypoxic area (P = 0.029), and had negative correlation with vessel density (P = 0.004). DCE-MRI measurements did not relate to either hypoxia or vessel density in U87MG tumors. Clinical data confirmed comparable signal changes in patients with glioblastoma. CONCLUSION: These data support further investigation of T1 -weighted OE-MRI to identify regional tumor hypoxia. The quantification of AUCOE has translational potential as a clinical biomarker of hypoxia.