Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha subunit but differ in their hormone-specific beta subunits. Site-directed mutagenesis was used to examine the role of the five disulfide bonds in the alpha subunit on the folding, assembly with the hCG beta subunit, and in cases where dimer formation occurred, receptor binding and signal transduction. Cysteine residues in the disulfide bonds formed by cysteines 7-31, 10-60, 28-82, 59-87, and 32-84 (Lapthorn, A., Harris, D. Littlejohn, A., Lustbader, J. Canfield, R., Machin, K., Morgan, F., and Isaacs, N. (1994) Nature 369, 455-461) were converted to alanine, and these mutants were transfected alone or together with the wild-type hCG beta gene into Chinese hamster ovary cells. The alpha Cys-10, 28, 60, 82, and 84 mutants were not secreted and in most cases were degraded at a faster rate than the native subunit. In addition, these mutants failed to assemble with the hCG beta subunit. Mutants with alterations at alpha Cys-7, 31, 32, 59, or 87 were secreted and combined with the beta subunit. Heterodimers containing a 7-31 double mutant bound to human lutropin-chorionic gonadotropin receptor expressed in transfected human fetal kidney cells, and stimulated cAMP comparable to wild-type hCG. Dimers containing the beta subunit with either single mutant alpha 59, alpha 87, alpha 32, or the alpha 59-87 double mutant showed much lower affinity for the receptor than wild-type hCG. These results suggest that disulfide bonds associated with alpha 7, alpha 31, alpha 59, alpha 87, and alpha 32 are not essential for the alpha subunit to fold into a form that will combine with the hCG beta subunit and to produce a biologically active dimer. This contrasts with observations of the hCG beta subunit where all the disulfide bonds are required for efficient combination and folding (Suganuma, N., Matzuk, M., and Boime, I. (1989) J. Biol. Chem. 264, 19302-19307). In addition, the lack of secretion of some mutants reflects previous observations that proteins which do not fold correctly are rapidly degraded. Thus, alpha subunit mutants which fold properly are secreted and can form heterodimers.