We had earlier shown that TGF-β controls proliferation, migration, and invasiveness of normal human trophoblast cells, whereas premalignant and malignant trophoblast cells are resistant to TGF-β. To identify signaling defects responsible for TGF-β resistance in premalignant and malignant trophoblasts, we have compared the expression of TGF-β signaling molecules in a normal trophoblast cell line (HTR-8), its premalignant derivative (RSVT2/C), and two choriocarcinoma cell lines (JAR and JEG-3). RT-PCR analysis revealed that all these cell lines expressed the mRNA of TGF-β1, -β2, and -β3, TGF-β receptors type I, II, and III, and post-receptor signaling genes smad2, smad3, smad4, smad6, and smad7 with the exception that TGF-β2 and smad3 were undetectable in JAR and JEG-3 cells. Immunoblot analysis confirmed the absence of smad3 protein in choriocarcinoma cells. Treatment with TGF-β1 induced smad3 phosphorylation and smad3 translocation to the nucleus in the normal and premalignant trophoblast cells. These results suggest that loss of smad3 may account for a functional disruption in the TGF-β signaling pathway in choriocarcinomas, but not in the premalignant trophoblast.