To correlate misonidazole concentrations and oxygen pressures (Po2) at identical locations within EMT6/Ro multi-cell spheroids (mean diameters ± SD: 867 ± 20 μm), Po2 measurements were performed with oxygen-sensitive microelectrodes during incubation of these spheroids with tritiated misonidazole (10 mg/l; 445 μCi/mg). In each individual spheroid, Po2 profiles were correlated with the corresponding spatial distribution of misonidazole as quantified by conventional autoradiography and grain counting. To compare the oxygenation status of spheroids in the measuring chamber with that of spheroids in spinner culture, misonidazole labeling was performed in both environments following the same protocol. All experiments were conducted in 20% oxygen and BME or in 5% oxygen and DMEM to obtain spheroids with different degrees of oxygenation. Labeled misonidazole was fairly evenly distributed in the outer, better oxygenated regions of EMT6 spheroids. In contrast, there was an accumulation of the labeled substance near central necrosis where low oxygen tensions were measured. Grain densities were similar at corresponding oxygen pressures under both environmental conditions. Except for some scatter, grain density as a function of oxygen pressure showed little variation in the Po2 range of 20–60 mm Hg, but exhibited a steep increase below 10 mm Hg. The findings imply that a substantial rise in local misonidazole labeling indicates a meta-bolically active tissue region at low Po2 that is not necessarily identical with the radiobiologically hypoxic cell fraction. A comparison of the labeling densities of spheroids in spinner flasks and in the Po2 measuring chamber indicates that oxygenation of spheroids is better in rotation culture than during microelectrode measurements. © 1995 Wiley-Liss, Inc.