Endothelial cell migration is an important process that occurs during embryonic vasculogenesis and angiogenesis, wound healing, and tumor growth and metastasis, and after denudation injury following angioplasty and bypass grafting. Mechanisms regulating this process involve a complex interplay of cytoskeletal reorganization, cell-cell adhesion, and cell-extracellular matrix interactions. Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is constitutively expressed in endothelial cells, leukocytes, monocytes, lymphocytes, and platelets. PECAM-1 undergoes dynamic dephosphorylation during endothelial cell migration in vitro and during vasculogenesis in the murine conceptus. In transfected 3T3 cells, in cultured endothelial cells, and in the murine conceptus undergoing vasculogenesis, the tyrosine phosphorylation state of PECAM-1 Y686 correlates with the migratory state of the cells. In the present study, we investigate the role of PECAM-1 in modulating endothelial cell migration in vitro using retroviral transduction of c-myc epitope-tagged, wild-type, Y686 to F-mutated PECAM-1 and extracellular domain-deleted PECAM-1 constructs in endothelial cells devoid of endogenous PECAM-1 (ECV304 cells) and in endothelial cells expressing high levels of endogenous PECAM-1 (bovine aortic endothelial cells, BAEC). In ECV304 cells, the expression of wild-type PECAM-1 inhibited migration rates, whereas in BAEC, overexpression of wild-type PECAM-1 had no effect. Expression of Y686 to F-mutated PECAM-1 or extracellular domain-deleted PECAM-1 in BAEC increased migration rates. These data support the concept that endothelial cell migration is modulated, in part, by a balance between PECAM-1 cytoplasmic domain-mediated "inside-out" and "outside-out" signaling resulting from PECAM-1-mediated cell-cell interactions and integrin-extracellular matrix interactions.