The human placenta is characterized by extensive trophoblast invasion of the uterus. Indeed, extravillous cytotrophoblast cells invade the decidua and the upper third of uterine spiral arteries in the myometrium. This invasion is reflected in situ by the expression of specific markers. In order to study this invasion process, we have established an in vitro culture model of human extravillous trophoblast isolated from first trimester chorionic villi. The aim of this study was to investigate the effect of a composite matrix, the Matrigel required for the culture of this homogenous population of extravillous trophoblasts (EVCT), on their in vitro differentiation. The effect of Matrigel was studied on different markers characterized by immunocytochemistry and by real-time polymerase chain reaction assay of transcripts. In addition, the expression of 12 different matrix metalloproteases and their inhibitors were investigated. We show that human extravillous cytotrophoblasts acquire an invasive phenotype on Matrigel associated with a specific pattern of protease gene expression. This in vitro model will be of interest to study the cellular mechanisms involved in abnormal trophoblast invasion observed in poor placentation and preeclampsia.