Tumor hypoxia is a persistent obstacle for traditional therapies in solid tumors. Strategies for mitigating the effects of hypoxic tumor cells have been developed under the assumption that chronically hypoxic tumor cells were the central cause of treatment resistance. In this study, we show that instabilities in tumor oxygenation are a prevalent characteristic of three tumor lines and previous characterization of tumor hypoxia as being primarily diffusion-limited does not accurately portray the tumor microenvironment. Phosphorescence lifetime imaging was used to measure fluctuations in vascular pO₂ in rat fibrosarcomas, 9L gliomas, and R3230 mammary adenocarcinomas grown in dorsal skin-fold window chambers (n = 6 for each tumor type) and imaged every 2.5 minutes for a duration of 60 to 90 minutes. O₂ delivery to tumors is constantly changing in all tumors, resulting in continuous reoxygenation events throughout the tumor. Vascular pO₂ maps show significant spatial heterogeneity at each time point, as well as between time points. The fluctuations in oxygenation occur with a common periodicity within and between tumors, suggesting a common mechanism, but have tumor type–dependent spatial patterns. The widespread presence of fluctuations in tumor oxygenation has broad ranging implications for tumor progression, stress response, and signal transduction, which are altered by oxygenation/reoxygenation events. [Cancer Res 2008;68(14):5812–9]