Previously we discovered that NPY induces ischemic angiogenesis by activating Y2 and Y5 receptors. The receptors that mediate specific steps of the complex process of angiogenesis are unknown. Here, we studied in vitro NPY receptors subtypes involved in migration, proliferation, and differentiation of human endothelial cells. In cells that expressed Y1, Y2, and Y5 receptors, NPY bimodally stimulated migration and proliferation with a 2‐fold increase at 10⁻¹² M and 10⁻⁸ M (high‐ and low‐affinity peaks, respectively). Preincubation of cells with NPY up‐regulated the Y5 receptor and markedly enhanced endothelial cell migration and proliferation. NPY‐induced endothelial cell migration was mimicked by agonists and fully blocked by antagonists for any specific NPY receptors (Y1, Y2, or Y5), while proliferation was blocked by any two antagonists (Y1+Y2, Y1+Y5, or Y2+Y5), and capillary tube formation on Matrigel was blocked by all three (Y1+Y2+Y5). Thus, NPY‐induced angiogenesis requires participation of Y1, Y2, and Y5 receptor subtypes, with the Y5 receptor acting as an enhancer. We propose that these receptors form heteromeric complexes, and the Y1/Y2/Y5 receptor oligomer may be the uncloned Y3 receptor.—Movafagh, S., Hobson, J. P., Spiegel, S., Kleinman, H. K., Zukowska, Z. Neuropeptide Y (NPY) induces migration, proliferation, and tube formation of endothelial cells bimodally via Y1, Y2, and Y5 receptors. FASEB J. 20, 1327–1337 (2006)