Although lutropin (LH) and follitropin (FSH) are synthesized in the same pituitary gonadotropes, their secretion patterns in response to several experimental paradigms are not the same. Previous studies showing differences in secretion kinetics and the magnitude of hormone release by secretagogues imply differences in mechanisms for the storage and release of these hormones. To examine the secretory fate of LH and FSH, the genes encoding the common α-subunit and the corresponding β-subunits were transfected in rat somatotrope-derived GH3 cells, which contain regulated and constitutive secretory pathways. The use of a gene transfer/heterologous cell system avoids physiological variations and functional heterogeneity of gonadotropes. Pulse-labeling and subsequent chase experiments demonstrated that although one third of newly synthesized FSH enters a regulated pathway, the majority is released constitutively. This contrasts with LH, which is mainly secreted through a regulated pathway. Although stored LH and FSH are released from GH3 cells in response to both KCl and forskolin, the magnitude of FSH release by secretagogues is smaller than that of LH. In Chinese hamster ovary cells, which are devoid of regulated secretory pathway and lack secretory granules, the mature forms of LH and FSH are neither stored nor released by secretagogues. These observations indicate that the intracellular mechanisms for the storage and release of LH and FSH differ and suggest that primary secretion of LH and FSH is via the regulated and constitutive pathways, respectively.