The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common α-subunit and a hormone-specific β-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the α-subunit sequences required for association with either β-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both β-subunits assembled with a single α-subunit. To address whether the α-subunit can interact with more than one β-subunit simultaneously, we genetically linked the FSHβ- and CGβ-subunit genes to the common α-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHβ-CGβ- α), 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 α-subunit shuttles between the two β-subunits during early stages of accumulation in the endoplasmic reticulum. 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 FSHβ- and CGβ-subunits and an α-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 α- and both CGβ- and FSHβ-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the α-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the α/β-contacts are not a pre-requisite for receptor recognition.