Trophoblast invasion of the human uterus is stringently controlled by the microenvironment. Invasive extravillous trophoblast cells in situ as well as in culture express a selective repertoire of cell surface integrins. Since migration is a necessary step in the invasion cascade, we tested whether certain integrins or invasion-regulating molecules, i.e., TGF-beta, IGF-II, and IGFBP-1 produced at the fetomaternal interface had a functional role on trophoblast migration. Flow cytometric analysis of integrin expression and the use of an in vitro cell migration assay revealed that exogenous TGF-beta upregulates integrin expression and reduces migratory ability to the invasive trophoblast, whereas IGF-II has no effect on integrin expression but stimulates migration. Trophoblast migration was inhibited in the presence of alpha 5 and beta 1 integrin blocking antibodies, indicating its dependence on the expression of these subunits. Furthermore, IGFBP-1, which contains an RGD sequence recognizing certain integrins, stimulated migration, an effect that was blocked by pretreatment with anti-alpha 5 or -beta 1 blocking Abs. These studies demonstrate that the migration of first trimester invasive trophoblast in vitro (1) requires the expression of alpha 5 and beta 1 integrin subunits, (2) is inhibited by TGF-beta, possibly due to increased cell adhesiveness to the extracellular matrix, (3) is stimulated by IGF-II by an as yet undetermined mechanism, and (4) is stimulated by IGFBP-1, likely by interaction with the RGD binding site of the alpha 5 beta 1 integrin. The invasion-regulating effects of TGF-beta, IGF-II, and IGFBP-1 may thus, at least in part, be due to their migration-regulating effects on the invasive trophoblast.