Tumor necrosis factor-alpha (TNF-alpha) in placenta is believed to be involved in pathogenesis of intrauterine growth restriction. In contrast, insulin-like growth factors (IGFs) are believed to be important for stimulation of fetal and placental growth. IGF-I stimulates metabolic and growth-promoting actions directly through its receptors: IGF-I receptor (IGF-IR), insulin receptor (IR) and IGF-I/insulin hybrid receptor (HR). However, it has not been elucidated which receptor mediates the growth promoting effects in fetal and placental growth. The current studies were undertaken to test whether TNF-alpha affects IGF-I action on placenta using human trophoblast cell cultures, and to test which receptor mediates growth promoting effects of IGF-I in placenta. Primary culture of trophoblast cells, which express IGF-IR, IR, and HR, were exposed to TNF-alpha, and the effects of IGF-I in stimulating trophoblast cell proliferation and migration were determined. Exposure to TNF-alpha attenuated the effects of IGF-I on cell proliferation and migration. To determine which receptors are involved in this inhibitory effect, the ability of IGF-I to stimulate phosphorylation of its receptors was analyzed in the presence of TNF-alpha. TNF-alpha exposure neither attenuated the phosphorylation of IGF-IR homodimer by IGF-I nor changed receptor abundance. In contrast, TNF-alpha reduced the ability of IGF-I to stimulate phosphorylation of HR with reducing amounts of HR. Exposure to TNF-alpha also attenuated phosphorylation of insulin receptor substrate-1 (IRS-1) and the association of IRS-1 with phosphatydilinositol-3 kinase (PI-3 kinase). Taken together, these findings indicate that TNF-alpha induces a loss of sensitivity to stimulation by IGF-I, through reducing amounts of HR and the stimulation of HR tyrosine kinase activity by IGF-I.