To elucidate the mechanism of secretory granule formation, we here identify the first intermediate in this process, the immature secretory granule, in the neuroendocrine cell line PC12 and demonstrate the packaging of a regulated secretory protein, secretogranin II, to immature secretory granules in a cell-free system. The formation of immature secretory granules was as fast (t1/2 approximately 5 min) as that of constitutive secretory vesicles identified by the presence of a rapidly secreted heparan sulfate proteoglycan. Using the cell-free system, the formation of post-Golgi secretory vesicles was found to be dependent upon ATP. Two distinct populations of vesicles were formed: immature secretory granules containing secretogranin II and constitutive secretory vesicles containing the heparan sulfate proteoglycan. These results show that in a cell-free system, a constitutive and a regulated secretory protein are sorted upon exit from the trans-Golgi network.