The glycoprotein hormone family are heterodimers, which include the placental hormone human chorionic gonadotropin and pituitary hormones follitropin, lutropin and thyrotropin . They are comprised of common alpha subunit and a hormone-specific beta subunit. Based on the chorionic gonadotropin crystal structure, it was suggested that the quaternary subunit interactions are crucial for biological activity. However, a number of observations using single chain analogs where the beta and alpha subunits are genetically linked (NH2-CGbeta-alpha; CGbetaalpha orientation), implied that the heterodimeric-like quaternary configuration is not a prerequisite for biological activity. To study 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 variant was constructed where the carboxyl terminus of alpha was fused to CGbeta amino terminus (NH2-alpha-CGbeta; alphaCGbeta orientation). The secretion rate of alphaCGbeta from transfected CHO cells was less than CGbetaalpha. Also, the alphaCGbeta tether was not recognized by dimer-specific monoclonal antibodies and did not bind to LH/CG receptor. To define if one or both subunit domains were modified in alphaCGbeta, it was co-transfected with the alpha or CGbeta gene. In each case, alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes were formed indicating chorionic gonadotropin dimer-specific epitopes were established. The alphaCGbeta/alpha complex bound to receptor indicating that the beta domain in the alphaCGbeta tether was still functional. In contrast, no significant receptor binding of alphaCGbeta/CGbeta was observed indicating a major perturbation in the a domain. These results suggest that although dimeric-like determinants are present in both alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes, the receptor binding determinants in the alpha 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.