Vascular endothelial growth factor (VEGF) has been identified as an endothelial cell-specific mitogen with potent angiogenic properties. VEGF is overexpressed in pathologic angiogenesis observed in tumor growth, rheumatoid arthritis, and retinal angiogenic diseases such as diabetic retinopathy and retinopathy of prematurity. VEGF expression in physiologic angiogenesis, i.e., vasculogenesis, has also been reported in the embryonic organs such as brain, kidney, spleen, and lung. However, the details of VEGF expression in vasculogenesis remain largely unclear. To determine if VEGF contributes to vasculogenesis in the developing tissues, VEGF expression was studied by both immunohistochemistry and in situ hybridization in newborn rat retinas on postnatal days 3, 7, 14, and 30. Vasculogenesis was assessed by both the ink perfusion method and a histologic examination. To identify the cell types of VEGF-expressing cells, immunohistochemistry for cell markers such as glial fibrillary acidic protein and von Willebrand factor was performed. On postnatal days 3 and 7, when retinal vasculogenesis was active, VEGF mRNA and protein(s) were prominently expressed in the ganglion cell and the inner nuclear layers. In rats, as well as humans, these two layers are where the retinal vessels develop, and these two layers depend solely on the retinal vessels. In addition to the ganglion and the inner nuclear layers, VEGF protein(s) were located in the endothelial cells of the developing vessels and the angioblasts, i.e., endothelial precursors. On postnatal day 14, when vasculogenesis became inactive, VEGF mRNA expression markedly decreased. These results indicated that VEGF expression in the developing retinas is temporally and spatially correlated with retinal vasculogenesis.