Hypoxia induces angiogenesis through the VEGF signaling pathway; however, signal propagation of VEGF under hypoxia is not understood fully. In this study, we examined alterations of VEGF signaling under hypoxia and its determinant in endothelial cells. To analyze VEGF signaling during hypoxia, human umbilical vein endothelial cells (HUVECs) were exposed to 3 h hypoxia (1% O₂), followed by 3 h reoxygenation or 12 h hypoxia. Hypoxia induced the expression of VEGF mRNA; however, it was not associated with an increase of tube formation by HUVECs. During 3 h hypoxia, VEGF-induced phosphorylation of VEGFR-2 and downstream molecules were significantly inhibited without a change of VEGFR-2 expression, but it was completely restored by reoxygenation. VEGF-mediated VEGFR-2 phosphorylation was associated with a reduction of cellular ATP under hypoxia (65.93 ± 8.32% of normoxia; mean ± s.e.m.; P<0.01). Interestingly, attenuation of VEGFR-2 phosphorylation was restored by the addition of ATP to prepared membranes from 3 h hypoxia-treated cells. In contrast with 3 h hypoxia, exposure of cells to 12 h hypoxia decreased VEGFR-2 expression and VEGF-mediated VEGFR-2 phosphorylation. The magnitude of VEGFR-2 phosphorylation was not fully restored by the addition of ATP to prepared membranes from cells exposed to 12 h hypoxia. These data indicate that ATP is an important determinant of VEGF signaling under hypoxia, and suggest that the activation process of VEGFR-2 was modified by sustained hypoxia. These observations contribute to our understanding of signal alterations of VEGF in endothelial cells under hypoxia.