The 9L rat gliosarcoma is a widely used experimental brain tumor which has an unusual radiation response. The radiation sensitivity of cells in subcutaneous tumors has been shown to be intermediate between those of aerobic and hypoxic 9L cells in vitro, and cells in spheroids and intracranial tumors appear to be as sensitive as fully aerobic cells. The hypoxic marker misonidazole was used to investigate the distribution of oxygen in these 9L systems. In vitro calibration of binding of [3H]-misonidazole as a function of oxygen concentration demonstrated an inverse relationship similar to those of several other experimental tumors. In autoradiograms of sections from tumors labeled in vivo the grain density rose gradually from the periphery of the tumor to the center. Over millimeter distances the distribution of grains was remarkably uniform, in contrast to the substantial variation reported for several other tumors. The grain density as a function of distance from capillaries was essentially constant. Several interpretations of this result are possible, including the postulate that intermittent blood flow occurs in all 9L tumor capillaries, which results in the majority of the binding of misonidazole occurring during the periods of transient hypoxia. Cells adjacent to the necrotic center in 9L spheroids and the rare necrotic regions in tumors appeared to be severely hypoxic, based on the quantity of misonidazole they bound. Spheroid "cure" experiments demonstrated that these cells were not clonogenic.