During placental development cytotrophoblast stem cells fuse to form the syncytiotrophoblast, a multinucleate cytoplasm with a brush border in contact with the maternal blood. Biochemical differentiation including the expression of placental-specific proteins and hormones accompanies this maturation. However, the biochemical mechanisms responsible for these events are unknown. We have defined a system in which single cytotrophoblast-like cells of the human choriocarcinoma (BeWo) cell line undergo fusion and extensive morphological differentiation following their treatment with effectors of cyclic AMP metabolism. Forskolin incubation caused a dose-dependent increase in intracellular and secreted cyclic AMP and a coordinate fusion of cells which yielded syncytia containing hundreds of nuclei per cytoplasm and a mature dense "placental-like" brush border. These fused cells also synthesized and secreted large amounts of both subunits of chorionic gonadotropin. However, they continued to synthesize several other placenta-specific proteins--placental-like alkaline phosphatase, placental lactogen, and SP1--at rates similar to those in control cells. Other reported effectors of cyclic AMP metabolism also induced cell fusion, although theophylline, an inhibitor of phosphodiesterase, induced fusion by a cyclic AMP-independent mechanism. Additionally, unlike the case with forskolin, treatment of BeWo cells with theophylline did not induce other morphological features of mature syncytiotrophoblasts. Thus, this system will allow one to examine the biochemical mechanism of placental cell fusion in the absence of other variables of cell differentiation.