Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common α-subunit but differ in their hormone-specific β-subunits. Both subunits have five and six disulfide bonds, respectively, which consist of cystine knot structure. It is evident from numerous studies that the structure of β-subunits is rigid, whereas that of α-subunit is flexible and can be molded by a β-subunit. Previously, we reported that secreted forms of α mutants where either cysteine residue in the disulfide bond 7-31 or 59-87 was converted to alanine contained a disulfide-linked homodimer in addition to a monomer. To study whether the hCGβ-subunit affects the conformations of α mutants, α-subunits lacking either the 7-31 or 59-87 disulfide bond were expressed with wild-type (WT) hCGβ in Chinese hamster ovary cells, and homodimer formation and glycosylation of dimerized α-subunit were assessed by continuous labeling with [³⁵S]methionine/cysteine, immunoprecipitation with anti-α or -hCGβ serum, digestion with endoglycosidase-H or -F, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. Our data showed that α homodimer was not observed in the half-Cys mutants except one, where cysteine at position 7 was converted to alanine, in the presence of β-subunit. This finding indicated that hCGβ-subunit rescued the α half-Cys mutants from the formation of intermolecular disulfide-linked homodimer by preferentially combining with the α mutants. In both free WT and all mutants treated with endoglycosidase-H, no or faint bands were recognized as the same migration as seen in endoglycosidase-F treatment. Even in the endoglycosidase-H sensitive cases, the amount of sensitive α-subunits was less than 5% of total α-subunits. In contrast to free α-subunits, distinct endoglycosidase-H sensitive bands were seen in both WT and mutants, although the ratio was various. We concluded that hCGβ-subunit affects the folding and glycosylation of the α-subunit mutants.