Human CG (hCG) is a member of the glycoprotein hormone family characterized by a heterodimeric structure consisting of a common α-subunit non-covalently bound to a hormone-specific β-subunit. The two subunits are highly intertwined and only the heterodimer is functional, implying that the quaternary structure is critical for biological activity. To assess the dependence of the bioactivity of hCG on the heterodimeric interactions, α- and β-subunits bearing mutations that prevent assembly were covalently linked to form a single chain hCG. Receptor binding and signal transduction of these analogs were tested and their structural integrity analyzed using a panel of monoclonal anti-bodies (mAbs). These included dimer-specific mAbs, which react with at least four different epitope sites on the hormone, and some that react only with the free β-subunit. We showed that there was significant loss of quaternary and tertiary structure in several regions of the molecule. This was most pronounced in single chains that had one of the disulfide bonds of the cystine knot disrupted in either the α- or β-subunit. Despite these structural changes, the in vitro receptor binding and signal transduction of the single chain analogs were comparable to those of the nonmutated single chain, demonstrating that not all of the quaternary configuration of the hormone is necessary for biological activity.