Purpose-EF5, a 2-nitroimidazole hypoxia marker, was used to study the presence, levels and prognostic significance of hypoxia in primary head and neck squamous cell tumors.Methods and Materials-22 patients with newly diagnosed squamous cell carcinoma of the oral cavity, oropharynx or larynx with at least 2 years of clinical follow-up were included in this study. Quantitative analysis of EF5 immunofluorescence was carried out and these data were compared to patient outcome.Results-EF5 immunostaining demonstrated substantial inter-tumoral hypoxic heterogeneity. The majority of cells in all tumors were well oxygenated. Three patterns of EF5 binding in cells were identified using criteria of the cellular region that was stained (peripheral or central) and the relationship of binding to necrosis. We tested the association between EF5 binding levels with eventfree and overall survival, irrespective of the pattern of cellular binding or treatment regime. Patients with tumors containing EF5 binding regions corresponding to severe hypoxia (≤0.1% oxygen) had a shorter event-free survival time than patients with pO 2 values >0.1% (p=0.032). Nodal status was also predictive for outcome.Conclusions-These data illustrate the potential utility of EF5 binding based on quantitative immunohistochemistry of tissue pO 2 and provide support for development of noninvasive hypoxia PET studies with 18 F-EF5.