Glycoprotein hormones have a common $alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $alpha$-subunit to the COOH-terminus of the $eta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${Delta}96$, Lys was substituted at position 95 to form ${Delta}95$, His was inserted at position 93 to form ${Delta}93$ and Tyr was substituted at position 87 to form ${Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${Delta}96$, ${Delta}95$, and ${Delta}93$ mutants were efficiently secreted into the medium but the ${Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${Delta}87$ mutant. However, the ${Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${Delta}95$ and ${Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $alpha$-subunit is very important for the secretion and functioning of this hormone.