To delineate the roles of O₂ and vascular endothelial growth factor (VEGF) in the process of angiogenesis from the embryonic aorta, we cultured mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) in a three-dimensional type I collagen gel matrix. During 8 days of culture under 5% O₂, but not room air, the addition of VEGF to explants stimulated the formation of CD31-positive, Flk-1-positive, Gs-IB₄-positive structures in a concentration-dependent manner. Electron microscopy showed the structures to be capillary-like. VEGF-induced capillary-like structure formation was inhibited by sequestration of VEGF via addition of soluble Flt-1 fusion protein or anti-VEGF antibodies. Expression of Flk-1, but not Flt-1, was increased in embryonic aorta cultured under 5% O₂ relative to room air. Our data suggest that low O₂ upregulates Flk-1 expression in embryonic aorta in vitro and renders it more responsive to VEGF.