Formation of the human placenta requires a subset of cytotrophoblast stem cells to acquire an invasive phenotype. We examined the effect on cytotrophoblast invasiveness of growth factors that control the differentiation of other cells. Exogenous TGF-β1 PDGF-AA PDGF-BB and TNF-α affected neither cell morphology nor the rate of cytotrophoblast invasion in vitro. In contrast addition of EGF to first trimester cytotrophoblast cultures produced dramatic changes in morphology and a severalfold increase in invasive capacity. The effects of EGF on later gestation cytotrophoblasts, whose invasive capacity is diminished were much less pronounced. Next we investigated whether cytotrophoblasts themselves produce ligands that interact with the EGF receptor. A radioimmunoassay and a radioreceptor assay failed to detect EGF receptor ligands in cytotrophoblast-conditioned medium. Likewise by RT-PCR cytotrophoblasts expressed neither EGF nor TGF-α mRNA. In contrast EGF receptor mRNA was expressed and its protein levels remained constant during the experiment. Immunolocalization using F(ab′) fragments of an anti-human EGF antibody failed to detect this growth factor in the chorionic villus. We conclude that maternal ligands that interact with the EGF receptor could play an important role by up-regulating trophoblast invasion particularly during the early stages of pregnancy.