Hypoxia inducible factors HIF1α and HIF2α are important proteins involved in the regulation of the transcription of a variety of genes related to erythropoiesis, glycolysis and angiogenesis. Hypoxic stimulation results in rapid increase of the HIF1α and 2α protein levels, as a consequence of a redox-sensitive stabilization. The HIFαs enter the nucleus, heterodimerize with the HIF1β protein, and bind to DNA at the hypoxia response elements (HREs) of target genes. In this study we evaluated the immunohistochemical expression of these proteins in 108 tissue samples from non-small-cell lung cancer (NSCLC) and in normal lung tissues. Both proteins showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells, tumoural vessels and tumour-infiltrating macrophages, as well as in areas of metaplasia, while normal lung components showed negative or very weak cytoplasmic staining. Positive HIF1α and HIF2α expression was noted in 68/108 (62%) and in 54/108 (50%) of cases respectively. Correlation analysis of HIF2α expression with HIF1α expression showed a significant association (P< 0.0001, r= 0.44). A strong association of the expression of both proteins with the angiogenic factors VEGF (P< 0.004), PD-ECGF (P< 0.003) and bFGF (P< 0.04) was noted. HIF1α correlated with the expression of bek-bFGF receptor expression (P= 0.01), while HIF2α was associated with intense VEGF/KDR-activated vascularization (P= 0.002). HIF2α protein was less frequently expressed in cases with a medium microvessel density (MVD); a high rate of expression was noted in cases with both low and high MVD (P= 0.006). Analysis of overall survival showed that HIF2α expression was related to poor outcome (P= 0.008), even in the group of patients with low MVD (P= 0.009). HIF1α expression was marginally associated with poor prognosis (P= 0.08). In multivariate analysis HIF2α expression was an independent prognostic indicator (P= 0.006, t-ratio 2.7). We conclude that HIF1α and HIF2α overexpression is a common event in NSCLC, which is related to the up-regulation of various angiogenic factors and with poor prognosis. Targeting the HIF pathway may prove of importance in the treatment of NSCLC.© 2001 Cancer Research Campaign http://www. bjcancer. com