OBJECTIVES To develop a method to isolate cells of human citotrophoblast and to assess its invading and differentiation capacity. TYPE OF STUDY Experimental biomedical. MATERIALS AND METHOD Citotrophoblasts of healthy placentas of full-term pregnancies were isolated by digestion with dispase and purification in a density gradient. The purity by immunoreactivity to citokeratin 7 and the invasiveness of the cells of citotrophoblast in Matrigel were evaluated. The enzymatic activity was determined through zimography and hCG secreted was quantified by means of ELISA. The expression of alpha 1 and alpha 5 integrins was evaluated by immunohistochemistry. RESULTS Citotrophoblasts with a purity of 97% were obtained; they differentiated themselves, in a spontaneous way, to a syncytium after four days. There was a growing production of hCG. Maximum invasiveness of citotrophoblasts ocurred the first two days, when their phenotype was mononuclear and coincided with the secretion of pro-MMP-9, and then it diminished according with the culture time. Immunoreactivity to the alpha 1 and alpha 5 integrins was observed in citotrophoblast cells with mononuclear phenotype. This immunoreactivity was lower in cells with phenotype of syncytium. CONCLUSIONS It was created an in vitro model that replicates events of the early development of the placenta. These events resemble the invasion and differentiation phase of the citotrophoblast. This model has potential utility in the study of the mechanisms of damage in preeclampsia.