Hypoxia is an important regulator of vascular endothelial growth factor (VEGF) expression, and VEGF is associated with poor prognosis in bladder cancer. To investigate further the mechanisms of VEGF regulation, we examined VEGF expression by mRNA and protein analysis in four human bladder cancer cell lines, showing a progression from well to poorly differentiated phenotypes under varying conditions of confluence and hypoxia (0.1% O(2)) and with chemical mimics of hypoxia. Hypoxia significantly increased VEGF protein expression in all cell lines, although this effect was dependent on the degree of confluence. The superficial bladder cancer cell line RT4 lost hypoxia inducibility at confluence, whereas inducibility was maintained in the invasive cell lines 253J and EJ28. This pattern of VEGF expression in the invasive cell lines correlated with the expression of the transcription factor hypoxia inducible factor-1 alpha (HIF-1 alpha) and with hypoxia-inducible factor-2 alpha (HIF-2 alpha) and in RT4 correlated with a marked reduction in HIF-1 alpha inducibility at confluence. Using the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY 294002, we show that this VEGF hypoxia-inducible pathway regulated by HIF-1 alpha is distinct from a PI 3-kinase-dependent pathway, which regulates basal amounts of VEGF, but does not affect inducibility. Both HIF-1 alpha and HIF-2 alpha protein and mRNA were up-regulated in primary human bladder tumors (n = 12) compared with normal bladder specimens (n = 4), with significant intertumor variation. These results suggest that components of the hypoxia response pathway, including HIF-1 alpha and HIF-2 alpha, are important cofactors in the regulation of VEGF in bladder cancer and are therapeutic targets in this disease.