In this study we show that decidua conditioned media (DCM) downregulate Connexin 40 (Cx40) expression in extravillous trophoblast (EVT) outgrowths and can promote EVT differentiation to the invasive phenotype resulting in switching of integrin and EGF receptor expression. This suggests that growth factors secreted by the decidua, such as EGF, mediate trophoblast migration/ invasion and may do so by modulating Cx40 expression and function. To test this hypothesis we have utilized migration assays using cell lines expressing Cx40. Migration assays were performed with Jeg-3, Jeg-3 overexpressing Cx40 (JpUHD) and JAR cells seeded on fibronectin-coated inserts with 8 mu m pores and incubated in the absence or presence of serum-starved decidual cells. Cell migration was only observed in the presence of DCM. Conversely overexpression of Cx40 in Jeg-3 cells resulted in inhibition of cell migration as Compared to wild-type control. Addition of DCM to Cultured JAR cells resulted in the downregulation of Cx40 protein. EGF is known to stimulate trophoblast migration/invasion and was detected in DCM therefore, we investigated the action of EGF on Cx40. EGF (10 ng/mL) resulted in the downregulation of Cx40 in the JAR cell line. However, EGF had no effect on JAR cell migration. We conclude that decidual secretion of growth factors, such as EGF, may act to prime trophoblast for migration/invasion through modulation of connexin expression and function.