It has been documented that hypoxia enhances coronary vasculogenesis and angiogenesis in cultured embryonic quail hearts via the upregulation of vascular endothelial growth factor (VEGF). In this study, we compared the functions of two VEGF splice variants. Ventricles from 6-day-old embryonic quail hearts were cultured on three-dimensional collagen gels. Recombinant human VEGF₁₂₁ or VEGF₁₆₅ were added to the culture medium for 48 h, and vascular growth was visualized by immunostaining with a quail-specific endothelial cell (EC) marker, QH1. VEGF₁₆₅ enhanced vascular growth in a dose-dependent manner: 5 ng/ml of VEGF₁₆₅ slightly increased the number of ECs, 10 ng/ml of VEGF₁₆₅ increased the incorporation of ECs into tubular structures, and at 20 ng/ml of VEGF₁₆₅ wider tubes were formed. This pattern plateaued at the 50 ng/ml dose. In contrast, VEGF₁₂₁ did not enhance either the number of ECs or tube formation at these or higher dosages. Combined effects of hypoxia and exogenous VEGF₁₆₅ were then compared. Tube formation from the heart explants treated with both hypoxia and 50 ng/ml of VEGF₁₆₅ had a morphology intermediate to those treated with hypoxia or VEGF₁₆₅ alone. Immunocytochemistry study revealed EC lumenization under all culture conditions. However, the addition of VEGF₁₆₅ stimulated the coalescence of ECs to form larger vessels. We conclude the following: 1) VEGF₁₂₁ and VEGF₁₆₅ induced by hypoxia have different functions on coronary vascular growth, 2) unknown factors induced by hypoxia can modify the effect of VEGF₁₆₅, and 3) EC lumenization observed in the heart explant culture closely mimics in vivo coronary vasculogenesis.