Oxygen diffusion distance was measured in solid tumor "cubes" prepared by excising the tumor from the mouse and incubating 1-2 mm sided tumor cubes in spinner culture flasks with fluorescent drugs (AF-2 or DM113) which bind to hypoxic cells. After incubation, frozen sections were prepared and examined for AF-2 or DM113 binding using a fluorescence image processing system. Alternatively, cubes were stained with a slowly penetrating fluorescent dye, Hoechst 33342, prior to disaggregation and measurement of AF-2 binding using flow cytometry. The distance from the cube surface to the AF-2 stained region (i.e., the thickness of the unstained rim) was used as an indication of oxygen diffusion distance, which depends on the square root of the tumor oxygen consumption rate. Oxygen diffusion distance was dependent on tumor type, external oxygen concentration, and temperature during incubation with AF-2, but was unaffected by tumor size up to 1.2 gm or position of the cube within the tumor. Oxygen diffusion distances (in planar geometry) measured for cubes prepared from SCCVII, RIF-1, Lewis lung or WiDr tumors were 107, 123, 153 and 193 microns, respectively. For SCCVII and WiDr tumors, the percentage of blood vessels separated by a distance greater than double the mean oxygen diffusion distance was used as an indication of the hypoxic fraction.