The human glycoprotein hormones CG, LH, FSH, and TSH are heterodimers composed of a common α subunit noncovalently associated with a hormone-specific β subunit. Recently, it was reported that a covalently fused triple-domain gonadotropin analog containing FSHβ, CGβ, and a subunits was dually active because it bound to both FSH and human CG (hCG)/LH receptors. However, it is not known whether both activities can be uncoupled from each other or whether they change in tandem when modifications are made in the molecule. To address this point, we constructed a triple-domain analog containing FSHβ, LHβ, and a subunits, and variants of this analog differing in the carboxyl-terminal region of LHβ. All of the analogs exhibited bifunctional action, i.e. they bound to both LH/hCG and human FSH receptors. FSH binding and signal transduction were similar for all variants and differed less than 2-fold from that of the heterodimer. In contrast, the triple-domain variants manifested distinct individual differences in LH activity. Binding affinity of the longest variant was 30-fold lower than that of the heterodimer. Shortening the length of the LHβ carboxyl-terminal region resulted in decreasing affinities between 210- and more than 480-fold. The potency of adenylate cyclase activation for LH/hCG also decreased as the carboxyl length of LHβ subunit decreased. Thus, while minimally affecting the FSH activity, truncating the carboxyl end of the LHβ subunit in the triple-domain analogs alters the alignment of the LHβ-α domains, presumably at the junction between the subunits, and perturbs epitopes required for receptor binding. These data imply that the relative potencies of the two gonadotropin components of a triple-domain structure are independent from each other and can be selectively modified. Because there is a strong rationale for FSHfLH combinations for clinical protocols and patients exhibit variations in metabolic responses in the ratio of FSH/LH, the ability to vary the individual activities represents a potential addition to the therapeutic repertoire for treating infertility.