Human trophoblast differentiates by the fusion of cytotrophoblasts to form syncytiotrophoblast. To determine factors controlling this process, the effects of epidermal growth factor (EGF) on trophoblast differentiation were studied using long term serum-free culture of isolated trophoblast. Only trophoblast was present in the cultures, as demonstrated by positive immunoperoxidase staining with beta hCG, cytokeratin, and trophoblast-specific H315 monoclonal antisera and by the absence of contaminating endothelial cells, fibroblasts, and macrophages, as shown by negative staining with vimentin and OKM1 monoclonal antisera. EGF induced large sustained increases in hCG and human placental lactogen (hPL) secretion in a dose-dependent manner. The minimum effective dose was 0.1 ng/mL, and the maximum effective dose was 1 ng/mL. Light and electron microscopic studies showed EGF-induced differentiation of cytotrophoblast to form syncytiotrophoblast. DNA content and cell number did not change during the process. The formation of syncytia thus probably accounted for the increase in hCG and hPL secretion. We conclude that EGF causes morphological differentiation, but not cell proliferation, of trophoblasts, and the differentiation results in increased hCG and hPL secretion from the syncytia.