It is well known that T(3) plays a crucial role in the maintenance of early pregnancy through the induction of endocrine function in villous trophoblasts. The effects of T(3) on extravillous trophoblast (EVT) function, however, remain to be elucidated. To investigate the possible role of T(3) in the regulation of EVT invasion to the decidua, we have examined whether T(3) affects EVT invasive potential and the expression of matrix metalloproteinase-2 (MMP-2), MMP-3, tissue inhibitor metalloproteinase-1, fetal fibronectin (FN), and integrin alpha(5)beta(1) in cultured early placental EVTs. Isolation and purification of trophoblasts differentiating into EVTs were performed by the enzymatic digestion of the anchoring chorionic villi, with the use of human FN-precoated culture dishes and FN-precoated Matrigel Transwells. The cells attached to the dishes were subcultured in DMEM supplemented with 10% fetal bovine serum for 48 h and were characterized by RT-PCR analysis after 24-h subculture and immunocytochemical analysis after 48-h subculture for specific EVT markers. Thereafter, the cultured cells were stepped down to a 4% fetal bovine serum condition and cultured in the presence or absence of T(3) (10(-8) m) for the subsequent 72 h. Matrigel invasion assay demonstrated that the treatment with T(3) significantly increased the number of cell projections of subsequent 24-, 48-, and 72-h cultured EVTs. RT-PCR analysis revealed that the treatment with T(3) increased the expression of MMP-2, MMP-3, fetal FN, and integrin alpha(5)beta(1) mRNA in subsequent 24-h cultured EVTs compared with those in control cultures. Immunocytochemical and Western immunoblot analyses revealed that treatment with T(3) increased the expression of MMP-2 and MMP-3 in subsequent 48-h cultured EVTs compared with those in control cultures. The present results suggest that T(3) (10(-8) m) may play a vital role in up-regulating the invasive potential of EVTs into the decidua.