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 identicalalpha subunit, and thus thebeta subunit contains determinants for sorting into the regulated pathway. Previously, we demonstrated that a hydrophobic carboxyl-terminal heptapeptide of the LHbeta subunit (Leu-Ser-Gly-Leu-Leu-Phe-Leu), not found in the FSHbeta 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 LHDeltaT (LHbeta subunit lacking the carboxyl-terminal seven amino acids) dimers in the rat somatotrope-derived GH(3) cell line in which both the regulated and constitutive secretory pathways operate. Pulse-chase labeling demonstrated that the LHDeltaT dimer was diverted to the constitutive pathway, resulting in a significant decrease in the corresponding intracellular pool. Forskolin stimulated LH dimer release 3-fold, which was accompanied by a parallel decrease of intracellular LH; only marginal forskolin stimulation of LHDeltaT was seen. Immunofluorescence after cycloheximide treatment demonstrated decreased retention of LHDeltaT compared with LH, consistent with increased constitutive secretion of LHDeltaT. We also demonstrated that fusing the heptapeptide to the carboxyl terminus of the FSHbeta 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.