The neuropilin-1 (np1) receptor binds the 165 amino-acid form of vascular endothelial growth factor165 (VEGF165) and functions as an enhancer that potentiates VEGF165 signaling via the VEGFR-2 tyrosine-kinase receptor. To study the mechanism by which neuropilins potentiate VEGF activity we produced a VEGF165 mutant (VEGF165KF) that binds to neuropilins but displays a much lower affinity toward VEGFR-1 and VEGFR-2. VEGF165KF failed to induce VEGFR-2 phosphorylation in cells lacking neuropilins. However, in the presence of np1, VEGF165KF bound weakly to VEGFR-2, induced VEGFR-2 phosphorylation, and activated ERK1/2. Interestingly, VEGF165KF did not promote formation of VEGFR-2/np1 complexes nor did high concentrations of VEGF165KF inhibit VEGF165 induced formation of such complexes, suggesting that VEGF165 does not stabilize VEGFR-2/np1 complexes by forming bridges spanning VEGFR-2 and np1. VEGF121 is a VEGF form that does not bind to neuropilins. Surprisingly, both np1 and neuropilin-2 (np2) enhanced VEGF121-induced phosphorylation of VEGFR-2 and VEGF121-induced proliferation of endothelial cells. The enhancement of VEGF121 activity by np1 was accompanied by a 10-fold increase in binding affinity to VEGFR-2 and was not associated with the formation of new VEGFR-2/np1 complexes. These observations suggest that neuropilins enhance the activity of VEGF forms that do not bind to neuropilins, indicate that np2 is a functional VEGF receptor, and imply that spontaneously formed VEGFR-2/np1 complexes suffice for efficient neuropilin mediated enhancement of VEGF activity.