Tumour hypoxia as driving force in tumour progression and treatment resistance has been well established. Assessment of oxygenation status of tumours may provide important prognostic information and improve selection of patients for treatment. In this study, a large homogenous group of 103 laryngeal carcinomas has been investigated in the presence of hypoxia by pimonidazole binding and the usefulness of Carbonic anhydrase IX (CA-IX) and vascular parameters as surrogate markers of hypoxia. These parameters are further related to clinical and biological characteristics. One hundred and three patients with T2-T4 larynx carcinoma were included. They were given the hypoxia marker pimonidazole intravenously (i.v.) 2h prior to taking a biopsy. Expression of all the parameters was examined by immunohistochemistry, excluding large necrotic areas. Among tumours a large variation in pimonidazole positivity (hypoxic fraction based on pimonidazole, HFpimo) (range 0-19%) and CA-IX expression (hypoxic fraction based on CA-IX staining, HFCA-IX) (range 0-34%) was observed. In 67% of the tumours, hypoxia involved 1% of the viable tumour area. HFpimo and HFCA-IX correlated significantly albeit weak (p=0.04). Both parameters showed weak inverse correlations with the relative vascular area (RVA) (p=0.01). HFpimo was further associated with histopathological grade, with poorly differentiated tumours being more hypoxic. The fraction of the tumour area positive for both pimonidazole and CA-IX correlated significantly with N stage. From these results, it was concluded that CA-IX and RVA have only limited value for measuring hypoxia and are not as robust as pimonidazole, probably due to the influence of other factors in the microenvironment. A combination of staining patterns of exogenous and endogenous markers might give important additive information about tumour biology and behaviour.