The crystal structure of human CG reveals that each subunit is a member of the superfamily of cystine-knot growth factors. Although the distribution of the cysteine residues in all the beta-subunits is conserved, the conformation of the human FSH dimer differs from that of the CG/LH dimers. This suggests that the function of the cystine bonded loops in the human FSHbeta-subunit may differ from that in the CGbeta-subunit. To address this issue, we deleted two disulfide bonds in the FSHbeta domain: cys 20-104 and cys 28-82, which correspond to the disulfide bonds 26-110 and 34-88, respectively, in the CGbeta-subunit. The cys 26-110 bond is associated with the "seat-belt" region and cys 34-88 is a bond in the cystine knot. Coexpression of the wild-type alpha-subunit with the FSHbeta cysteine mutants in CHO cells revealed no detectable heterodimer. The FSHbeta mutants were then incorporated into a single chain where the beta-subunit is genetically fused to the alpha-subunit. In such a model, the rate-limiting subunit assembly step is by-passed and mutations that otherwise block heterodimer formation can be evaluated in terms of biological activity. Compared with the nonmutated single chain, the single-chain 28-82 mutant is secreted more slowly and its recovery is substantially reduced, whereas secretion and recovery of the 20-104 mutant was not significantly affected. The receptor binding affinity of the cys 28-82 mutant did not differ from wild-type and binding of the cys 20-104 mutant was decreased only 2-fold. The signal transduction data parallel the binding affinities, although the maximal accumulation of cAMP is less for the cys 20-104 mutant than that seen for cys 28-82 and nonmutated single-chains variants. These data support the hypothesis that the determinants for intracellular behavior and bioactivity of the gonadotropins are not the same, and that the cystine knot is a critical determinant for the formation of a stable, assembly-competent subunit. In addition, the data imply that the "seat-belt" conformation does not play a prominent role in the bioactivity of FSH.