Trophoblast is the major functional cell type of the placenta. The purpose of this study was to devise a means to isolate trophoblast cells from the monkey placenta and to examine their capacity to differentiate in vitro. Methods originally devised for the isolation of human cytotrophoblast cells produced poor yields and a low degree of purity when applied to the near-term rhesus monkey placenta. However, a procedure has been developed which allows the isolation of a cell population consisting of more than 95% cytotrophoblast based on intermediate filament immunocytochemistry. The cells sedimented between densities of 1.040 and 1.053 g/ml on continuous Percoll density gradient centrifugation. When maintained in culture the cells adhered and formed aggregates of mononuclear cells by 24 h. By 5 d in culture, immunofluorescent staining using antidesmoplakin and antinuclear antibodies revealed that most colonies consisted of large multinucleated masses similar to syncytiotrophoblast. These results demonstrate trophoblast cells from monkey placental villi can be isolated with a high degree of purity and undergo morphologic differentiation in vitro. This preparation should enable investigators to study many functional characteristics of these cells throughout gestation.