First-trimester human placental villi were cultured on 3H-leucine-labeled extracellular matrices isolated from the PF HR9 and PYS-2 cell lines. Both cell lines produced an extracellular matrix that contained basement membrane-specific macromolecules, including type IV collagen, laminin and proteoglycan. Both matrices promoted outgrowth of cells from the villi which, according to morphological criteria, were identified as cytotrophoblastic cells. As the cells migrated from the attachment site, they caused a marked focal dissolution of the matrix which was accompanied by a concomitant release of 3H-labeled material into the media. Approximately half of this material chromatographed near the inclusion volume of Sephadex G-50, indicating that the labeled matrix components had been degraded. This phenomenon was dependent on the age of the placenta. Second-trimester placental villi also adhered to the matrix, but no areas of dissolution were formed and no significant amounts of radioactivity were released into the medium. These results suggest that culture of first-trimester human placental villi on extracellular matrices may be useful for the study of some of the early embryonic events leading to human implantation, during which the trophoblastic cells erode the uterine epithelium.