We demonstrate a novel approach to measuring regional tumor oxygen tension using 1 9 F pulse burst saturation recovery echo planar imaging (EPI) relaxometry of hexafluorobenzene. Hexafluorobenzene offers exceptional sensitivity to changes in oxygen tension, and has a single resonance making it ideal for imaging studies. By combining a pulse burst saturation recovery preparation sequence with EPI, the relaxation experiments were performed in ∼20 min facilitating measurements of dynamic changes in pO 2 accompanying interventions. Direct intratumoral administration of hexafluorobenzene permitted labeling of specific regions of interest, and imaging provided maps of pO 2 , confirming distinct intra tumoral heterogeneity. For a group of three Dunning prostate adenocarcinoma R3327-AT1 tumors interrogation of the central tumor region showed skewed pO 2 distributions with considerable radiobiological hypoxia (∼90% voxels had pO 2 < 15 torr) when rats breathed 33% O 2 . Altering the inspired gas to pure oxygen caused distributions to shift towards increased pO 2 with significant increases in mean oxygen tension (p < 0.05) in two cases. Interrogation of both central and peripheral regions in a fourth tumor showed bimodal distribution for tumor oxygenation including ∼75% voxels with pO 2 > 15 torr. EPI allows the fate of individual voxels to be traced: upon altering the inspired gas to pure oxygen those voxels with baseline pO 2 > 30 torr showed significant changes (p < 0.05), whereas those with pO 2 < 16 torr showed minimal response. The precision of the measurements, together with the ability to simultaneously examine dynamic changes in multiple regions should provide a useful technique for investigating tumor hypoxia with respect to therapy.