Although synthesized in the same pituitary gonadotropes, the secretion profiles of lutropin (LH) and follitropin (FSH) differ. LH is secreted through a regulated pathway and associated with a bolus release at mid estrous cycle. In contrast, the majority of FSH is secreted constitutively with an incremental increase until ovulation. Both share an identical subunit and thus, the subunit contains determinants for sorting into the regulated pathway. Previously, we demonstrated that a hydrophobic carboxyl terminal heptapeptide of the LH subunit (Leu-Ser-Gly-Leu-Leu-Phe-Leu), not found in the FSH subunit, influences the intracellular behavior of the LH dimer. To test the hypothesis that the peptide contributes to differential sorting, we monitored the fates of LH and LHT (LH subunit lacking the carboxyl terminal seven amino acids) dimers in the rat somatotrope-derived GH3 cell line in which both the regulated and constitutive secretory pathways operate. Pulse-chase labeling demonstrated that the LHT dimer was diverted to the constitutive pathway, resulting in a significant decrease in the corresponding intracellular pool. Forskolin stimulated LH dimer release three-fold, which was accompanied by a parallel decrease of intracellular LH, and only marginal forskolin stimulation of LHT was seen. Immunofluorescence after cycloheximide treatment demonstrated decreased retention of LHT compared with LH, consistent with increased constitutive secretion of LHT. We also demonstrated that fusing the heptapeptide to the carboxyl terminus of the FSH subunit resulted in an increased regulated secretion of this FSH analog compared with wild-type FSH. These data are the first to identify a novel structural determinant responsible for the sorting of a member of the glycoprotein hormone family into the regulated secretory pathway.