It is well established that hypoxia potently stimulates tumor angiogenesis by activating hypoxia inducible factor-1 (HIF-1)-induced proangiogenic factors, such as vascular endothelial growth factor. However, very little is known about the role of hypoxia in incipient angiogenesis in avascular tumors during their early stages of growth. To noninvasively investigate the functional significance of hypoxia and HIF-1 activation in incipient tumor angiogenesis, we genetically engineered HCT116 human colon carcinoma cells and 4T1 mouse mammary carcinoma cells with constitutively expressed red fluorescence protein as a tumor marker and green fluorescence protein (GFP) as a reporter for hypoxia and HIF-1 activation. The accuracy of GFP fluorescence in reporting hypoxia was confirmed by flow cytometry analysis and by immunohistochemical comparison with pimonidazole, a well-established hypoxia marker drug. Mouse dorsal skin-fold window chambers showed that incipient angiogenesis preceded a detectable level of hypoxia. The detectable levels of hypoxia were spatially and temporally related with more intensive secondary angiogenesis following the initial onset of new vessel formation. Selective killing of hypoxic cells by tirapazamine efficiently eliminated or delayed the detection of hypoxic cells, but it did not significantly delay the onset of incipient angiogenesis. These findings provide the first in vivo evidence that incipient tumor angiogenesis may not depend on hypoxia or HIF-1 activation. This is in contrast to the clear role of hypoxia in driving angiogenesis once initial tumor microvessel formation has occurred.