Cell adhesion molecule L1 was implicated in angiogenic processes, tumor formation and metastasis. Here, we provide evidence that the sixth Ig-like domain of L1 (L1Ig6) interacts with alpha(v)beta3 to induce process extension of human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis in vivo. HUVECs formed network-like structures on full-length L1 or L1Ig6 substrates comparable to structures found on matrigel. In the presence of mab alpha(v)beta3 or cyclic RGD, apoptosis was induced. In fibrin matrices where L1Ig6 was covalently incorporated, HUVECs formed multicellular and hollow processes through interactions between cell-surface alpha(v)beta3 and RGD-sites of matrix-immobilized L1Ig6. No such processes were induced by L1Ig6 having non-functional RDG-sites, or in the presence of mab alpha(v)beta3 or cyclic RGD. In those matrices, increased apoptosis was found. Co-immunoprecipitation of L1 or L1Ig6 with alpha(v)beta3 suggests close interactions. Furthermore, L1Ig6 stimulated HUVECs showed increased tyrosine phosphorylation of alpha(v)beta3 and phosphorylation of MAP kinases (ERK1 and ERK2) but not AKT indicating specific activation of alpha(v) and alpha(v)beta3 followed by activation of downstream kinases. Application of L1Ig6-modified fibrin matrices on CAMs induced 50-60% increased alpha(v) and alpha(v)beta3 protein expression and in vivo angiogenesis indicated by approximately 50% increased mean vascular length density. The results demonstrate angiogenic potential of L1Ig6 involving ligation and activation of alpha(v)beta3.