Recruitment of vascular smooth muscle cells (SMC) by endothelial cells (EC) is essential for angiogenesis. Endothelial-derived heparin binding EGF-like growth factor (HB-EGF) was shown to mediate this process by signaling via ErbB1 and ErbB2 receptors in SMCs. 1) Analysis of ErbB-ligands demonstrated that primary ECs expressed only HB-EGF and neuregulin-1. 2) Primary SMCs expressed ErbB1 and ErbB2, but not ErbB3 or ErbB4. 3) Consistent with their known receptor specificities, recombinant HB-EGF, but not neuregulin-1, stimulated tyrosine phosphorylation of ErbB1 and ErbB2 and migration in SMCs. 4) Neutralization of HB-EGF or inhibition of ErbB1 or ErbB2 blocked 70-90% of the potential of ECs to stimulate SMC migration. Moreover, 5) angiopoietin-1, an EC effector with a role in recruitment of SMC-like cells to vascular structures in vivo, enhanced EC-stimulated SMC migration by a mechanism involving up-regulation of endothelial HB-EGF. Finally, 6) immunohistochemical analysis of developing human tissues demonstrated that HB-EGF was expressed in vivo in ECs associated with SMCs or pericytes but not in ECs of the hyaloid vessels not associated with SMCs. These results suggest an important role for HB-EGF and ErbB receptors in the recruitment of SMCs by ECs and elaborate on the mechanism by which angiopoietins exert their vascular effects.