The role of integrins in the processes of adhesion and migration makes them attractive potential participants in the complex events of embryo implantation and placentation. Recently, the role of the alpha(v)beta(3)-integrin pathway was shown in the insulin-like growth factor-I (IGF-I)-stimulated migration of extravillous trophoblast (EVT) cells. This study was designed to investigate the role of alpha(5)beta(1)-integrin in this respect. Using cultured EVT cells, migration assays were carried out for IGF-I-treated or untreated cells in the presence or absence of the GRGDSP and GRGESP hexapeptides, alphaIR3, and a blocking antibody against alpha(5)beta(1)-integrin. Immuno-electron microscopy and immunofluorescent staining were performed to localize the distribution of alpha(5)beta(1)- and alpha(v)beta(3)-integrins, Rab5a, paxillin, phospho-FAK (pFAK), and vinculin. The results showed that IGF-I-induced migration of EVT cells was abolished following treatment with GRGDSP hexapeptide, alphaIR3, and a blocking antibody against alpha(5)beta(1)-integrin. Further, statistical analysis showed that the area-related numerical density of the alpha(5)beta(1)-integrin in the perinuclear regions was significantly higher than in the cell extensions. Immunocytochemical experiments demonstrated an up-regulation in internalization rate of alpha(5)beta(1)-integrin in IGF-I-stimulated EVT cells. Furthermore, alpha(5)beta(1)-integrin exhibited co-localization with Rab5a, but not with alpha(v)beta(3)-integrin, pFAK, paxillin, and vinculin at the focal adhesions of the EVT cells. Taken together, these findings suggest an essential role for alpha(5)beta(1)-integrin in IGF-I-promoted migration of EVT cells. It is possible therefore that IGF-I-induced internalization of alpha(5)beta(1)-integrin may be an important event during the migration of EVT cells in the complex processes of implantation and placentation.