The biochemical characterization of human yolk-sac tumor (YST) antigen 2G10, detected by monoclonal antibody (MAb) M912-2G10, was studied. Previous results indicated that glycolipids having a non-reducing terminal N-acetyllactosamine structure were the epitope of 2G10 on human erythrocytes. In this study, the glycoprotein nature of 2G10 on the infantile embryonal carcinoma line, MTE, was investigated. 2G10 activity, measured by a new enzyme-linked immunosorbent assay (2G10-ELISA), was recovered in residual fractions of MTE from which glycolipids were removed. Chromatographically, 3H-galactose-labelled 2G10 on MTE had a molecular weight (mw) of about 580 kDa, which decreased after pronase or alkaline-borohydride treatment. Our results indicate the glycoprotein nature of 2G10 on MTE. Furthermore, 2G10, both on erythrocytes and on MTE, was sensitive to galactosidase but not to neuraminidase and fucosidase, suggesting that terminal galactose is involved in the antigenic structure. It was also found by 2G10-ELISA that 2G10 sheds from tumor cells. Shedding occurs in nude mice transplanted with MTE as well as in patients with germ-cell tumors (GCTs). The serum level of 2G10 in non-tumor patients was low, but high levels were detected in patients with YSTs and with GCTs having YST components. Immunohistochemically, the presence of 2G10-positive YST components was shown in patients who had high serum levels of 2G10. Sera from other urogenital and childhood solid tumors did not have elevated 2G10. The mw of shed 2G10 was lower than that of 2G10 on the cell surfaces. Our results clearly indicate the usefulness of serum 2G10 as a tumor marker for GCTs having YST components.