The presence of radiation-resistant hypoxic cells in some solid tumors is known to predict for relapse after radiotherapy. Use of an endogenous marker of hypoxia would be a convenient alternative to current methods that measure tumor oxygenation, provided the marker could be shown to reliably identify viable, radiation-resistant, hypoxic cells. Carbonic anhydrase 9 (CA9) is a transmembrane protein overexpressed in a wide variety of tumor types and induced by hypoxia. Using a monoclonal antibody and cell sorting, CA9-positive cells in SiHa cervical carcinoma xenografts growing in immunodeficient mice were found to be clonogenic, resistant to killing by ionizing radiation, and preferentially able to bind the hypoxia marker pimonidazole. CA9 and pimonidazole immunostaining were compared in formalin-fixed sections from tumors of 18 patients undergoing treatment for cancer of the cervix. Excellent colocalization was observed, although the area of the tumor section that bound anti-CA9 antibodies represented double the number of cells that bound anti-pimonidazole antibodies. Occasional regions staining with pimonidazole but not CA9 could be indicative of transient changes in tumor perfusion. Results support the hypothesis that CA9 is a useful endogenous marker of tumor hypoxia.