An efficient noninvasive method for in vivo imaging of tumor oxygenation by using a low-field magnetic resonance scanner and a paramagnetic contrast agent is described. The methodology is based on Overhauser enhanced magnetic resonance imaging (OMRI), a functional imaging technique. OMRI experiments were performed on tumor-bearing mice (squamous cell carcinoma) by i.v. administration of the contrast agent Oxo63 (a highly derivatized triarylmethyl radical) at nontoxic doses in the range of 2–7 mmol/kg either as a bolus or as a continuous infusion. Spatially resolved pO₂ (oxygen concentration) images from OMRI experiments of tumor-bearing mice exhibited heterogeneous oxygenation profiles and revealed regions of hypoxia in tumors (<10 mmHg; 1 mmHg = 133 Pa). Oxygenation of tumors was enhanced on carbogen (95% O₂/5% CO₂) inhalation. The pO₂ measurements from OMRI were found to be in agreement with those obtained by independent polarographic measurements using a pO₂ Eppendorf electrode. This work illustrates that anatomically coregistered pO₂ maps of tumors can be readily obtained by combining the good anatomical resolution of water proton-based MRI, and the superior pO₂ sensitivity of EPR. OMRI affords the opportunity to perform noninvasive and repeated pO₂ measurements of the same animal with useful spatial (≈1 mm) and temporal (2 min) resolution, making this method a powerful imaging modality for small animal research to understand tumor physiology and potentially for human applications.