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In culture the human teratocarcinoma cell line HT-H generates both adherent monolayer and free-floating aggregates. Some populations of aggregated cells develop further to form cystic bodies. A previous study showed the morphological resemblance of the cystic bodies to cells of blastocyst of preimplantation embryo. In this study, HT-H adherent cells were further separated into two subpopulations, fast adhering and slow adhering cells. Fast adhering cells produce fibronectin, spread well onto substratum, and do not proliferate. In contrast, slow adhering cells do not produce fibronectin. Trophoblastic markers were examined in each morphological stage of HT-H cells and the following results were obtained. Only fast adhering cells produce progesterone. Human chorionic gonadotropin was secreted preferentially by fast adhering cells, about six times less by slow adhering cells, and was not secreted by aggregates or cystic bodies. All stages of HT-H cells express c-fos but only fast adhering cells express c-fms oncogene. Cytokeratin 18 was expressed in all stages of HT-H cells. The level of cytokeratin 18 is modestly decreased from adherent to aggregates further into cystic bodies. These results indicate that HT-H cells share properties with cells in trophoblast, placenta, and extraembryonic endoderm. Spontaneous differentiation of HT-H cultures results in the appearance of fast adhering cells which exhibit biochemical properties expected for syncytiotrophoblast.