Chronic tissue hypoxia may play a role in the pathogenesis of late radiation fibrosis. In order to investigate this hypothesis, the immunohistochemical distribution of pimonidazole hydrochloride (n = 14 patients) and carbonic anhydrase IX (CAIX) (n = 38 patients) was studied in samples of previously irradiated normal human tissue. One sample of irradiated breast tissue, which also showed marked histological features of radiation injury, stained positive for pimonidazole hydrochloride. No CAIX staining was seen in irradiated tissue other than some evidence of physiological hypoxia in the epidermis of two samples of irradiated skin; both were positive for pimonidazole and one was focally positive for CAIX. Pimonidazole hydrochloride staining of tissue with morphological changes of radiation injury could support a role for hypoxia in the pathogenesis of late normal tissue fibrosis in humans.