We have earlier shown that migration and invasiveness of first trimester human extravillous trophoblast cells are stimulated by IGF-II, independently of IGF type 1 receptor and that migration stimulation is the primary reason for increased extravillous trophoblast cell invasiveness induced by IGF-II. In the present study we examined the functional role of IGF type II receptor in IGF-II stimulation of extravillous trophoblast cell migration and the underlying signal transduction pathways including the participation of inhibitory G protein(s) and MAPK. The migratory ability of a well characterized in vitro propagated human first trimester extravillous trophoblast cell line expressing the phenotype of extravillous trophoblast cells in situ was quantitated with a Transwell migration assay under different experimental conditions. We found that the extravillous trophoblast cells expressed an abundance of IGF type 2 receptor as detected by immunostaining and Western blots, and recombinant human IGF-II promoted their migration in a dose- and time-dependent manner. Both polyclonal and monoclonal IGF type 2 receptor-blocking antibodies blocked migration-stimulating effects of IGF-II. Two synthetic IGF-II analogs ([Leu27]IGF-II, which can bind to IGF type 2 receptor and IGF-binding proteins, but not IGF type 1 receptor, and [QAYL-Leu27]IGF-II, which can bind to IGFR-II, but neither IGFR-I nor IGF-binding proteins) both stimulated extravillous trophoblast cell migration to levels higher than those induced by wild-type IGF-II. These results reveal that IGF-II action was mediated by IGF type 2 receptor, independently of IGF type 1 receptor and IGF-binding proteins. Treatment of extravillous trophoblast cell membrane preparations with IGF-II decreased adenylyl cyclase activity in a concentration-dependant manner, indicating the participation of inhibitory G proteins in IGF-II action. This was substantiated further with the findings that increasing intracellular cAMP using forskolin or (Bu)2cAMP inhibited basal extravillous trophoblast cell migration and blocked IGF-II stimulation of migration. IGF-II treatment rapidly stimulated phosphorylation of MAPK (ERK-1 and -2), which was blocked by pretreatment of extravillous trophoblast cells with the MAPK kinase (MEK) inhibitor PD98059. Treatment with this inhibitor also blocked extravillous trophoblast cell migration in the presence or absence of IGF-II. These results, taken together, reveal that IGF-II stimulates extravillous trophoblast cell migration by signaling through IGF type 2 receptor, involving inhibitory G proteins and activating the MAPK pathway.