This study tested the hypothesis that selective blockade of neuropeptide Y (NPY) receptors Y1 and/or Y2 will affect the embryonic formation of coronary vessels induced by VEGF using an in vitro heart explant model. The ventricular apices from the hearts of C57BL/6 mouse embryos (E13) were placed on collagen I gel and cultured for 96 hrs in medium with 10% FBS. For the next 72 hrs the explants were cultured in medium with 1% FBS and treated with the Y receptor selective antagonists (10 nmol/μL) in the presence of NPY (1 nmol/μL) and/or VEGF (50 ng/mL). The explants were stained with Alexa Fluor 594-labeled GS-I Isolectine B4 and the total length of endothelial outgrowths (TLO) was quantified using Image-Pro Plus software. In each explant TLO was normalized by the explant perimeter and finally TLO of experimental explants was expressed as a percentage of TLO in control (VEGF-treated) explants. The VEGF-induced formation of endothelial outgrowths was not modified by stimulation of either Y receptor. However, selective blockade of Y2 receptors, but not of Y1 receptors, showed significant reduction in formation of VEGF-induced cord-like endothelial structures by ~68% (P<0.001). In addition, Y2 receptor blockade seemed to decrease the number of endothelial cells within the area surrounding the explants. Our data demonstrates that the NPY/Y2 signaling system plays a modulatory role in VEGF-induced coronary vessel formation.