The human choriocarcinoma cell line, BeWo, synthesizes the glycoprotein hormone, human chorionic gonadotropin (hCG). We have undertaken this study to compare the synthesized and secreted forms of hCG and their alpha- and beta-subunits in cell cultures of BeWo cells to those forms of normal placental cells by immunobinding techniques. BeWo cells appeared to synthesize and secrete one species of the respective hCG subunit. The immature alpha- and beta-subunits, synthesized in BeWo cells as well as those of placental cells, were digested by endoglycosidase H indicating N-linked sugar chain(s) to be the high-mannose type. The mature alpha- and beta-subunits, secreted by BeWo cells as well as subunits of urinary hCG which are usually used as a standard hCG secreted by normal placental cells, were sensitive to neuraminidase treatment indicating that these subunits have terminal sialic acid(s). Contrary to placental cells, mature subunits of BeWo hCG could not be found in any subcellular fraction indicating a rapid secretion rate or supporting the hypothesis that BeWo cells secrete hCG subunits without the formation of secretory granules. The alpha-subunit synthesized in BeWo cells had a slightly lower molecular weight than that of placental cells; however, the alpha-subunit secreted by BeWo cells had a slightly higher molecular weight than the alpha-subunit of urinary hCG. The beta-subunits synthesized and secreted by BeWo cells had slightly higher molecular weights than beta-subunits of both placental cells and urinary hCG. Even after digestion by N-glycanase as well as endoglycosidase H, molecular weights were still different between the respective subunits of BeWo and placental cells indicating that the apoprotein structures of BeWo hCG subunits may differ from those of placental cells. Moreover, urinary beta-subunit was sensitive to endo-alpha-N-acetylgalactosaminidase treatment but the beta-subunit secreted by BeWo cells was not, indicating that the structure of O-linked sugar chain(s), if present, may be unusual. Analysis of assembled and free forms of subunits of BeWo cell cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by immunobinding methods revealed that subunits are associated intracellularly and then secreted to the media as hCG. Moreover, only free beta-subunits, but not alpha-subunits, of BeWo hCG were found intra- and extracellularly.