The presence and significance of tumor hypoxia has been recognized since the 1950's. Hypoxic cells in vitro and in animal tumors in vivo are documented to be three times more resistant to radiation-induced killing compared to aerobic cells. There is now evidence that tumor hypoxia is treatment-limiting in many human cancers. One common way to describe the extent of hypoxia in individual and groups of tumors is the "hypoxic fraction." This measurement infers that cells are present in only two radiobiologically significant states: oxygenated and hypoxic. In this paper, we demonstrate the qualitative and quantitative presence of hypoxic tumor cells using the oxygen dependent metabolism of the 2-nitroimidazole, EF5. Two assumptions concerning the calculation and interpretation of the hypoxic fraction are considered. The first is the use of multiple animals to describe the radiation response at a given radiation dose. We hypothesize that the presence of intertumor variability in radiation response due to hypoxia could negatively influenced the characterization of the change in slope required to calculate the hypoxic fraction. The studies presented herein demonstrate heterogeneity of radioresponse due to hypoxic fraction within and between tumor lines. The 9L subcutaneous tumor studied in air-breathing rats demonstrates a 2 log variation in surviving fraction at 17 Gy. The Morris 7777 hepatoma, in contrast, showed little variability of radiation response. Our second question addresses the limitations of using the "hypoxic fraction" to describe the radiation response of a tumor. This calculated value infers that radiobiological hypoxia is a binary measurement: that a tumor contains two cell populations, aerobic cells with maximal radiosensitivity and hypoxic cells with maximal radioresistance. The classic work of Thomlinson and Gray, however, implies the presence of an oxygen gradient from tumors vessel through the tissues. In both the 9L and Q7 tumors, flow cytometric analysis of EF5 binding demonstrates a continuous range of cellular pO2 levels. These studies suggest that: 1) there is extensive intertumor variability of radiation response in certain tumor lines; 2) the variability in radiation response between individual tumors in a group may affect the ability to describe a particular tumor type's "hypoxic fraction" and 3) The oxygen status of tumor cells is a continuum. This realization affects the ability to apply a binary concept such as the "hypoxic fraction" effectively in radiobiology.