Tumor oxygenation has long been recognized as a significant factor influencing cancer therapy. We recently established a novel magnetic resonance in vivo approach to measuring regional tumor oxygen tension, FREDOM (Fluorocarbon Relaxometry Using Echo Planar Imaging for Dynamic Oxygen Mapping), using hexafluorobenzene (HFB) as the reporter molecule. We have now investigated oxygen dynamics in the two Dunning prostate R3327 rat tumor sublines, AT1 and H. FREDOM revealed considerable intratumoral heterogeneity in the distribution of pO(2) values in both sublines. The anaplastic faster-growing AT1 tumors were more hypoxic compared with the size-matched, well-differentiated, and slower-growing H tumors. Respiratory challenge with oxygen produced significant increases in mean and median pO(2) in all the H tumors (P<.001), but no response in half of the larger AT1 tumors (>3 cm(3)). Immunohistochemical studies using the hypoxia marker, pimonidazole, and the vascular endothelial cell marker, CD31, confirmed that the H tumors had more extensive vasculature and less hypoxia than the AT1 tumors. These results further validate the utilization of FREDOM to monitor tumor oxygenation and concur with the hypothesis that the level of hypoxia is related to tumor growth rate and poor vascularity.