The glycoprotein hormone family represents a class of heterodimers, which include the placental hormone human chorionic gonadotropin (CG) and the anterior pituitary hormones follitropin, lutropin, and thyrotropin. They are composed of common α subunit and a hormone-specific β subunit. Based on the CG crystal structure, it was suggested that the quaternary subunit interactions are crucial for biological activity. However, recent observations using single chain glycoprotein hormone analogs, where the β and α subunits are linked (NH2-CGβ-α; CGβα orientation), implied that the heterodimeric-like quaternary configuration is not a prerequisite for receptor binding/signal transduction. To study the heterodimeric alignment of the two subunit domains in a single chain and its role in the intracellular behavior and biological action of the hormone, a single chain CG variant was constructed in which the carboxyl terminus of α was fused to the CGβ amino terminus (NH2-α -CGβ; αCGβ orientation). The secretion rate of αCGβ from transfected Chinese hamster ovary cells was less than that seen for CGβα. The αCGβ tether was not recognized by dimer-specific monoclonal antibodies and did not bind to lutropin/CG receptor. To define if one or both subunit domains were modified in αCGβ, it was co-transfected with a monomeric α or CGβ gene. In each case, αCGβ/α and αCGβ/CGβ complexes were formed indicating that CG dimer-specific epitopes were established. The αCGβ/α complex bound to receptor indicating that the β domain in the αCGβ tether was still functional. In contrast, no significant receptor binding of αCGβ/CGβ was observed indicating a major perturbation in the α domain. These results suggest that although dimeric-like determinants are present in both αCGβ /α and αCGβ/CGβ complexes, the receptor binding determinants in the α domain of the tether are absent. These results show that generating heterodimeric determinants do not necessarily result in a bioactive molecule. Our data also indicate that the determinants for biological activity are distinct from those associated with intracellular behavior.