One of the most sensitive hypoxia detection methods is based on the observation that binding of nitroimidazoles to cellular macromolecules occurs as a result of hypoxia-dependent bioreduction by cellular nitroreductases. Nitroimidazole-binding techniques provide measurements of hypoxia to virtually any degree of spatial resolution and with a multiplicity of techniques. This paper demonstrates hypoxia imaging using in vivo EF5 binding with detection by a fluorochrome-conjugated monoclonal antibody. We investigated these techniques in the 9L glioma tumour, in part because the exact nature of the hypoxia in this tumour system is controversial. Our results demonstrate that following intravenous injection of EF5, binding and detection using a monoclonal antibody in 9L gliomas is specific and oxygen dependent. Detection of binding using fluorescence microscopy can be performed on frozen tissues; tissue sections can be counterstained with haematoxylin and eosin for light microscopic analysis. Alternatively, the distribution of hypoxia in a tumour can be inferred by examining individual tumour cells using flow cytometric techniques. Based upon the results presented herein, the radiation-resistant phenotype of 9L epigastric tumours grown in our laboratories can be associated with the presence of hypoxic cells.