Chorionic gonadotropin (CG) and lutropin (LH) are members of a family of glycoprotein hormones that share a common α subunit but differ in their hormone-specific β subunits. The glycoprotein hormone β subunits share a high degree of amino acid homology that is most evident for the LHβ and CGβ subunits having >80% sequence similarity. However, transfection studies have shown that human CGβ and α can be secreted as monomers and can combine efficiently to form dimer, whereas secretion and assembly of human LHβ is less efficient. To determine which specific regions of the LHβ and CGβ subunits are responsible for these differences, mutant and chimeric LHβ-CGβ genes were constructed and transfected into CHO cells. Expression of these subunits showed that both the hydrophobic carboxy-terminal seven amino acids and amino acids Trp8, Ile15, Met42, and Asp77 together inhibit the secretion of LHβ. The carboxy-terminal amino acids, along with Trp8, Ile15, Met42, and Thr58 are implicated in the delayed assembly of LHβ. These unique features of LHβ may also play an important role in pituitary intracellular events and may be responsible for the differential glycosylation and sorting of LH and FSH in gonadotrophs.