AbstractAngiogenesis plays critical roles in organ development during embryonic and fetal life, wound healing and in a variety of pathological conditions. Vascular endothelial growth factor (VEGF) is a secreted growth factor specific for vascular endothelial cells which induces angiogenesisin vivo. To gain a better understanding of the physiological role of VEGF, we have generated and characterized four murine monoclonal antibodies (mAbs) using the 165 amino acid species of recombinant human VEGF as immunogen. These mAbs (A3.13.1, A4.6.1, B4.3.1 and B2.6.2) belong to IgG1 isotype and have high affinities for VEGF (dissociation constants range from 2.2x10−8to 4x10−10M). Two different epitopes were detected with these mAbs. One epitope is recognized by mAbs A3.13.1 and B2.6.2, and the other recognized by mAbs A4.6.1 and B4.3.1. The epitope recognized by mAb A4.6.1 appears to be continuous while mAb B2.6.2 recognizes a discontinuous epitope. MAb A4.6.1 recognized three species of VEGF generated by alternative splicing, VEGF121, VEGF165and VEGF189while mAb B2.6.2 binds only VEGF165and VEGF189. Results using anin vitrobovine adrenal cortex endothelial cell proliferation assay, inin vivovascular permeability assay and anin vivoembryonic chicken angiogenesis assay showed that mAb A4.6.1 has potent VEGF neutralizing activities. MAb A4.6.1 was shown to block the binding of VEGF to its receptor(s) suggesting the inhibitory mechanism for VEGF activities.These well-defined mAbs should be very powerful tools to understand the structure-function relationship of various domains of VEGF and may have therapeutic potential.