Although the LHbeta- and chorionic gonadotropin-beta- (CGbeta) subunits share a high degree of sequence identity (>85%) in the first 114 amino acids, there is considerable sequence divergence at their carboxy ends. The CGbeta-subunit terminates with a unique carboxyl-terminal extension (115-145; carboxyl-terminal peptide), which contains four O-linked oligosaccharides, whereas the LHbeta-subunit bears a hydrophobic heptapeptide (115-121) at its carboxy terminus. LH is released through the regulated pathway in the pituitary, whereas CG is secreted constitutively from the placenta. We previously demonstrated in rat somatotroph-derived GH(3) cells that the LH is associated primarily with a regulated routing, and although the majority of CG was released constitutively from the cells, there was a fraction that was segregated through the regulated pathway. Moreover, we showed that the LHbeta heptapeptide is a determinant for the regulated secretion of LH. Given that the primary evolutionary change between LHbeta and CGbeta occurred at the carboxy terminus, these data suggested that the presence of the CGbeta carboxyl-terminal peptide region is responsible for the constitutive secretion of CG. A CG114 mutant (CGDeltaT) was constructed and expressed in GH(3) cells. Steady-state labeling and pulse-chase experiments demonstrated that the CGDeltaT entered the regulated pathway resulting in over 4-fold increase in the intracellular pool. The secretagogue, forskolin, stimulated CGDeltaT release over 3-fold, which was accompanied by a parallel intracellular decrease, and only marginal stimulation of CG was seen. Immunofluorescence demonstrated a unique membrane pattern of staining for CGDeltaT compared with dispersed cytoplasmic puncta for CG. Stimulation with forskolin caused a significant reduction in the relative fluorescence of CGDeltaT cells compared with a minor reduction for CG. These data show that the CGDeltaT analog resembles LH in its intracellular trafficking, further supporting the hypothesis that determinants at the carboxyl-terminal end of the CGbeta-subunit evolved from the LHbeta-subunit primarily to overcome the slow release and intracellular storage of LH resulting in rapid secretion of CG from the placenta.