In pregnancy tissues, oncofetal fibronectin (onfFN) has been localized specifically to the extracellular matrix (ECM) surrounding extravillous anchoring trophoblasts of the placental-uterine junction and chorion. When isolated from first or third trimester placentas, human cytotrophoblasts in culture secrete and deposit onfFN in the ECM. In addition, onfFN synthesis is significantly up-regulated in response to serum stimulatory factor(s). The goal of this study was to examine the role of transforming growth factor-beta (TGF beta), a cytokine present in uterine decidua, as a stimulator of trophoblast onfFN production. Our initial insight into the significance of TGF beta resulted from the serendipitous use of cord serum from a neonate with severe alloimmune thrombocytopenia. Trophoblasts cultured in medium containing this serum underwent normal morphological differentiation, but produced markedly less onfFN. In an analogous fashion, trophoblasts cultured in normal serum preincubated with anti-TGF beta neutralizing antibodies also produced significantly less onfFN. Exogenously added TGF beta 1 restored the ability of trophoblasts to produce onfFN by a factor of 4- to 5-fold in medium containing thrombocytopenic serum. In platelet-poor serum derived from human or bovine plasma, TGF beta 1 also induced onfFN synthesis, as assayed both in the conditioned medium and by immunocytochemical localization of onfFN in cell-associated ECM fibrils. Dose-response analysis demonstrated that the onfFN stimulatory response is sensitive to TGF beta, with an ED50 of 0.1-0.2 ng/ml. In a reciprocal fashion, TGF beta inhibited beta hCG secretion 3- to 4-fold. Our results demonstrate that TGF beta is a significant stimulator of trophoblast onfFN production. Furthermore, TGF beta appears to modulate trophoblast differentiation by up-regulating the expression of an anchoring trophoblast marker (onfFN) and down-regulating a phenotypic marker of villous syncytiotrophoblast (hCG beta). We speculate that trophoblast responsiveness to TGF beta in the implantation milieu contributes to trophoblast adhesion by stimulating the production of a trophoblast-derived implantation site fibronectin.