Endogenous hypoxia markers have been studied as prognostic indicators because they appear to be associated with tumor aggressiveness. This study was undertaken to compare the expression of two endogenous hypoxia markers, Hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase IX (CA IX), with regard to their prognostic significance. We also compared spatial distribution of HIF-1α and CA IX and examined their relationship with expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9, which may be regulated by hypoxia. We studied 74 resected stage I/II non-small cell lung cancers (NSCLCs) for expression of HIF-1α, CA IX, VEGF, and MMP-9 by immunohistochemistry, and the extent of tumor necrosis. Univariate and multivariate analyses were performed to assess prognostic implications of these markers for disease free survival. HIF-1α expression was strongly correlated with CA IX (r=0.667, p<0.001) and was co-localized with CA IX in corresponding areas. HIF-1α and CA IX expression were higher in areas with moderate to severe tumor necrosis relative to areas with minimal necrosis, suggesting their relationship with hypoxia. VEGF expression also showed a modest relationship with HIF-1α (p=0.07); however, there was no relationship between HIF-1α and MMP-9 expression (p>0.99). Expression of HIF-1α and CA IX above the median value was significantly associated with shorter disease free survival in univariate analysis (p<0.05). However, only high CA IX expression and pathologic stage were independent prognostic indicators in a multivariate analysis. Of the markers considered in this study, CA IX expression status was the most reliable hypoxia marker for predicting tumor aggressiveness.