Maternal IGFs regulate cytotrophoblast proliferation and, thereby, placental growth and function. IGF bioavailability is controlled by IGF-binding proteins (IGFBPs); in placenta, IGFBP-3 is particularly abundant. In other systems, IGFBP-3 can regulate cellular events independently of IGFs; these effects are thought to be mediated by TGFbeta receptors (TbetaR). We have examined IGFBP-3 regulation of IGF-dependent and -independent cytotrophoblast proliferation in first-trimester placental explants and the role of TbetaRII in mediating these effects. In the presence of IGFBP-3 (50 nm), IGF-induced (10 nm) proliferation (monitored by immunohistochemical analysis of Ki67 expression and bromodeoxyuridine incorporation) was significantly reduced (P < 0.05). IGFBP-3 also reduced basal proliferation independently of IGF receptor signaling. Immunohistochemical analysis demonstrated that TGFbeta signaling molecules [TGFbeta receptor I (TbetaRI), TbetaRII, TbetaRV, Smad-2, and ERK] are expressed in syncytium and/or cytotrophoblast. TGFbeta1 (10 ng/ml) enhanced cytotrophoblast proliferation and activated both Smad-2 and ERK-1/2, whereas IGFBP-3 activated only Smad-2. The function of both TGFbeta1 and IGFBP-3 was attenuated by a TbetaRII function-blocking antibody and by small interfering RNA-mediated knockdown of TbetaRII (P < 0.05); this was accompanied by a reduction in Smad-2 activation. This study demonstrates that both TGFbeta1 and IGFBP-3 signal through TbetaRI/II to influence human cytotrophoblast proliferation. However, downstream pathways are distinct, because IGFBP-3 acts only through Smad-2, whereas TGFbeta1 also phosphorylates ERK, resulting in opposite effects on cytotrophoblast proliferation. The effects of maternal growth signals on placental growth and function therefore depend on the balance of ligands, receptors, and signaling molecules at the syncytiotrophoblast surface. Therapeutic manipulation of this balance might offer a strategy to optimize placental development and pregnancy outcome.