The objectives of this research were to determine optimal conditions for culturing trophoblast cells in vitro and to ascertain whether cells maintained under these conditions are an adequate source of material for fetal chromosome studies. Best results were obtained with the use of 1.25 × 106 cells in a total volume of 4 ml of M-199 medium that contained 20% calf serum and 1 mg/ml glucose, incubated in an atmosphere of 95% air and 5% carbon dioxide. Cytogenetic analysis was performed in trophoblast cultures 14 to 64 days old. In all cases it was possible to identify cells in metaphase which were adequate for karotyping. Evidence that the cultured cells were fetal in origin was provided by two cases in which chromosome abnormalities-46,XY,inv(18), and 47,XX,+21-were identified both in the placental cultures and in leukocytes and fibroblasts from the fetus. The cytogenetic analysis was correct in the prediction of the fetal sex in seven cases; in two cases the prediction was questionable, and in two cases it was in error. The conclusion is that the use of trophoblast cells for fetal cytogenetic studies is not practical because of (1) difficulties in culturing the cells, (2) the low mitotic frequency of the cells in culture, and (3) contamination of the culture with maternal fibroblasts.