The human glycoprotein hormones chorionic gonadotropin (CG), thyrotropin (thyrotropin), lutropin (LH), and follitropin (FSH) are heterodimers composed of a common subunit and a hormone-specific subunit. The subunits assemble non-covalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs) and several of the subunit sequences required for association with either subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo i.e. both subunits assembled with a single subunit. To address if the subunit can interact with more than one subunit simultaneously, we genetically linked the FSH and CG subunit genes to the common subunit, resulting in a single chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple domain variant (FSH-CG-), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the subunit shuttles between the two subunits during early stages of accumulation in the ER. Therefore, constructs were engineered that would direct the synthesis of single chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple domain single chain chimeras contain the FSH and CG subunits and an subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that despite disrupting the intersubunit interactions between the and both CG and FSH subunits, these mutated analogs exhibit both activities in vivo comparable to non-mutated triple domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g., hormone-specific post-translational modifications, but when heterodimers are present in the circulation the / contacts are not a prerequisite for receptor recognition.