Vascular endothelial growth factor (VEGF), a potent angiogenic factor, uses two receptor tyrosine kinases, FLK/KDR and FLT, to mediate its activities. We have cross-linked 125I-VEGF165 to the cell surface of various tumor cell lines and of human umbilical vein endothelial cells. High molecular mass (220 and 240 kDa) and/or lower molecular mass (165 and 175 kDa) labeled complexes were detected depending on the cell type. The 220- and 240-kDa labeled complexes were shown to contain FLT and FLK/KDR receptors, respectively. On the other hand, the 165- and 175-kDa complexes did not seem to contain FLK/KDR or FLT but instead appeared to contain novel VEGF receptors with relatively low molecular masses of approximately 120 and 130 kDa. These receptors were further characterized in breast cancer MDA MB 231 cells (231), which did not form the high molecular mass complexes and which did not express detectable amounts of flk/kdr or flt mRNA. The 231 cells displayed one VEGF165 binding site, with a Kd of 2.8 x 10(-10) M and 0.95 1.1 x 10(5) binding sites per cell. By comparison, human umbilical vein endothelial cells had two binding sites, one with a Kd of 7.5 x 10(-12) M, presumably FLK/KDR, and the other with a Kd of 2 x 10(-10) M, a value similar to the VEGF binding sites on 231 cells. These lower affinity/molecular mass receptors on 231 cells cross-linked 125I-VEGF165 but not 125I-VEGF121. Accordingly, exon 7 of VEGF, which encodes the 44 amino acids present in VEGF165 that are absent in VEGF121, was fused to glutathione S-transferase (GST). The GST-VEGF-exon 7 fusion protein bound to heparin-Sepharose with a similar affinity as VEGF165 and inhibited the binding of 125I-VEGF165 to 231 cells. Cross-linking of 125I-GST-VEGF-exon 7 to 231 cells resulted in the formation of 150- and 160-kDa labeled complexes that presumably contained the 120- and 130-kDa lower affinity/molecular mass VEGF165 receptors. It was concluded that certain tumor-derived cell lines express novel surface-associated receptors that selectively bind VEGF165 via the exon 7-encoded domain, which is absent in VEGF121.