Endothelial Per-ARNT-Sim (PAS) domain protein-1 (EPAS-1)/hypoxia-inducible factor-2α (HIF-2α) is a member of the basic helix-loop-helix/PAS domain protein family and is considered to be an endothelial-specific, hypoxia-inducible transcription factor. Because hypoxia is a fundamental element of tumor biology determining clinical outcome, we performed an immunohistochemical study of EPAS-1 expression in a cohort of bladder cancer cases and assessed the possible correlation of EPAS-1 expression with tumor hypoxia and growth. In the 67 cases (37 radical cystectomy and 30 transurethral resection) studied, overexpression of EPAS-1/HIF-2α protein was not found in cancer cells or in normal tissues but was mostly found in stroma around cancer cells, and strong positive staining was noted in perinecrotic regions. The perinecrotic/tumorous expression of EPAS-1/HIF-2α was correlated statistically with higher histological grade (P < 0.001), advanced pathological T stage (P < 0.001), and presence of necrosis (P < 0.001). A parallel immunohistochemical analysis of a marker gene of vascular endothelial growth factor demonstrated its positive correlation with tumor grade, stage, and EPAS-1/HIF-2α overexpression, supporting the correlation of EPAS-1/HIF-2α up-regulation with tumor angiogenesis. To further clarify the relationship between hypoxia and vascularity in the perinecrotic/tumorous area with EPAS-1/HIF-2α expression, tissue microvessel density (MVD) was assessed. No significant correlation (P = 0.442) was found between EPAS-1/HIF-2α expression and MVD if the 67 tumors of different stages were all included. However, EPAS-1/HIF-2α-positive cases had lower MVD than EPAS-1/HIF-2α-negative cases (P = 0.001) if only invasive cancer cases were analyzed. In addition, in all EPAS-1/HIF-2α-positive staining cases, EPAS-1/HIF-2α-positive foci had lower MVD than EPAS-1/HIF-2α-negative foci (P < 0.001). Finally, using serial sections, the location of EPAS-1/HIF 2α expression was identified mainly in tumor-associated macrophage (TAM) as well as in some fibroblast cells. Focal TAM infiltration was identified at a higher level in EPAS-1-positive cases than EPAS-1-negative cases (P < 0.001). This is the first clinical report suggesting that hypoxia-induced, perinecrotic EPAS-1/HIF-2α expression is correlated with tumor progression and angiogenesis at higher grade and stage through focal TAM infiltration in invasive bladder cancer.