Preclinical development of therapeutic agents against cancer could greatly benefit from noninvasive markers of tumor killing. Potentially, the intracellular partial pressure of oxygen (pO2) can be used as an early marker of antitumor efficacy. Here, the feasibility of measuring intracellular pO2 of central nervous system glioma cells in vivo using 19F magnetic resonance techniques is examined. Rat 9L glioma cells were labeled with perfluoro-15-crown-5-ether ex vivo and then implanted into the rat striatum. 19F MRI was used to visualize tumor location in vivo. The mean 19F T1 of the implanted cells was measured using localized, single-voxel spectroscopy. The intracellular pO2 in tumor cells was determined from an in vitro calibration curve. The basal pO2 of 9L cells (day 3) was determined to be 45.3 ± 5 mmHg (n = 6). Rats were then treated with a 1× LD10 dose of bischloroethylnitrosourea intravenously and changes in intracellular pO2 were monitored. The pO2 increased significantly (P = 0.042, paired T-test) to 141.8 ± 3 mmHg within 18 h after bischloroethylnitrosourea treatment (day 4) and remained elevated (165 ± 24 mmHg) for at least 72 h (day 6). Intracellular localization of the perfluoro-15-crown-5-ether emulsion in 9L cells before and after bischloroethylnitrosourea treatment was confirmed by histological examination and fluorescence microscopy. Overall, noninvasive 19F magnetic resonance techniques may provide a valuable preclinical tool for monitoring therapeutic response against central nervous system or other deep-seated tumors. Magn Reson Med, 2010. © 2010 Wiley-Liss, Inc.