One of the major structural differences between the LHβ and CGβ subunits is the carboxy-terminal region: beyond amino acid 114, LHβ has a hydrophobic heptapeptide stretch, while CGβ contains a 31-amino acid hydrophilic carboxy-terminal peptide (CTP) that is O-glycosylated. The CGβ subunit is secreted quantitatively as a monomer and assembles efficiently whereas secretion and assembly of LHβ is inefficient. We previously implicated the carboxy-terminal heptapeptide as a determinant for the different intracellular behavior manifested by the LHβ subunit compared with the CGβ subunit. Here we tested the function of the heptapeptide and CTP domains by fusing them to their counterparts at amino acid 114 of CGβ or LHβ subunits. The secretion and assembly of these chimeras were examined in transfected Chinese hamster ovary cells. Removal of the heptapeptide enhanced the amount of LHβ subunit secreted 4-fold compared with intact LHβ. Fusion of this heptapeptide to CGβ114, i.e. CGβ lacking the CTP, decreased the amount of secreted subunit 2-fold compared with wild type human CGβ. Similar experiments reveal that although deleting the CTP from the CGβ subunit did not significantly alter secretion, the combination efficiency of the truncated subunit was reduced to 60%. Perturbing the native carboxy-terminal sequence of either subunit increased the heterogeneity of the secreted forms. This result suggests that these regions are also involved in the posttranslational processing of the asparagine-linked oligosaccharides of the β-subunits. Fusion of the LHβ heptapeptide to the truncated CGβ subunit decreased combination with the α-subunit. These data further support the hypothesis that the carboxy-terminal regions of LHβ and CGβ subunits play a role in the intracellular behavior of the corresponding heterodimers.