Hypoxia stabilizes HIF-1α (Hypoxia Inducible Factor-1α), which then triggers the expression of several genes involved in many aspects of cancer progression, including metabolic adaptation, cell survival and angiogenesis. The aim of our study was to evaluate the impact of HIF-1α and CA IX (carbonic anhydrase IX) (one of its target genes) expression on prognosis and treatment outcome of patients with breast cancer. Because of the extreme O2-dependent instability of the protein, we first validated HIF-1α staining using xenograft tumours that were subjected to experimental conditions mimicking surgical clamping or sitting at room temperature under normoxic conditions after surgical excision but before fixation. Afterwards, the immunohistochemical staining of HIF-1α and CA IX was evaluated in 132 invasive breast carcinomas with a 10-year follow-up, and correlated to classical clinicopathological parameters and response to adjuvant therapy. No significant correlation was found between tumour size or nodal status and the expression of HIF-1α or CA IX. Statistically significant association was found between HIF-1α or CA IX staining and the grade, hormonal receptors loss and the presence of carcinoma in situ. Overexpression of HIF-1α and CA IX correlates with a poor prognosis in breast cancer. We show that HIF-1α is an independent prognostic factor for distant metastasis-free survival and disease-free survival in multivariate analysis. Furthermore, overexpression of HIF-1α or CA IX correlates with a poor outcome after conventional adjuvant therapy. CA IX is, however, a weaker prognostic and predictive factor than HIF-1α, and its association with HIF-1α does not modify the survival curve neither response to therapy, compared to HIF-1α alone. © 2006 Wiley-Liss, Inc.