We established trophoblast cell cultures with extended lifespans by introducing into first trimester human trophoblasts the gene encoding simian virus 40 large T antigen. The transfected trophoblasts were characterized according to their expression of various morphological and functional markers. Both parental (HTR-8) and transfected (HTR-8/SVneo) lines were morphologically similar and positive for cytokeratin, confirming their epithelial (trophoblastic) identity. Whereas the parental cells senesced after 12-14 passages, the transfectants have been in culture for over 32 passages. Human chorionic gonadotrophin was detected only in the HTR-8/SVneo cells and not in the parental cells. Both lines required at least 5% serum in order to sustain growth in vitro and responded to transforming growth factor-beta (TGF-beta) with reduced [3H]-thymidine incorporation in a dose-dependent manner. Treatment with TGF-beta also resulted in decreased secretion of plasminogen activators (PAs) and reduced PA activity by both lines. Both cell lines secreted mostly 72-kDa type IV collagenase as determined by substrate gel zymography, but the level of secretion of this enzyme was not significantly affected by TGF-beta in either line. Even though both lines exhibited similar in vitro invasive abilities, only the invasiveness of the parental cells was reduced by TGF-beta. Neither parental or transfected cells were capable of growth in soft agar and no sign of tumor formation was evident more than 5 months after subcutaneous inoculation of the transfected cells into nude mice. These results indicate that apart from their ability to sustain prolonged growth in culture, the transfected HTR-8/SVneo cells share a number of phenotypic properties with the parental trophoblast cells. For this reason, these transfected trophoblasts may prove to be an important tool for the study of placental function and/or tumor progression.