The glycoprotein hormones lutropin (LH) and chorionic gonadotropin (CG) share a common structure consisting of an identical α subunit noncovalently linked to a hormone-specific β subunit. While LH is produced in the anterior pituitary, CG is synthesized in placenta. To compare the assembly, processing, and secretion of human LH and CG in the same cell type, we have expressed their subunits, individually and together, in mouse C-127 mammary tumor cells. Analysis of transfected clones revealed an unexpected difference in the secretion of individually expressed subunits. Whereas α and CGβ subunits were rapidly and quantitatively secreted, only 10% of newly synthesized LHβ subunit reached the medium. The remaining subunit was found in an intracellular, endoglycosidase H (endo H)-sensitive pool that had a turnover rate of ~8 h. Coexpression with α subunit resulted in 'rescue' of LHβ subunit by formation of LH dimer, which was efficiently secreted. However, combination of LHβ with α was slow, with an overall efficiency of only 50% despite the presence of excess α. In contrast, CGβ was rapidly assembled with the α subunit after synthesis. The two β subunits also differed in their influence on the N-linked oligosaccharide processing of combined α. The oligosaccharides of LH dimer were endo H resistant, while those of CG dimer reamined partially endo H sensitive. Thus, despite a high degree of homology between LHβ and CGβ, the two subunits differ in their secretion as free subunits, their rate of assembly with α subunit, and in their effect on the N-linked oligosaccharide processing of combined α.