Neuropeptide precursors synthesized at the rough endoplasmic reticulum are transported and sorted at the trans-Golgi network (TGN) to the granules of the regulated secretory pathway (RSP) of neuroendocrine cells. They are then processed into active peptides and stored in large dense-core granules (LDCGs) until secreted upon stimulation. We have studied the regulation of biogenesis of the LDCGs and the mechanism by which neuropeptide precursors, such as pro-opiomelanocortin (POMC), are sorted into these LDCGs of the RSP in neuroendocrine and endocrine cells. We provide evidence that chromogranin A (CgA), one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells, plays an important role in the regulation of LDCG biogenesis. Specific depletion of CgA expression by antisense RNAs in PC12 cells led to a profound loss of secretory granule formation. Exogenously expressed POMC was neither stored nor secreted in a regulated manner in these CgA-deficient PC12 cells. Overexpression of CgA in a CgA- and LDCG-deficient endocrine cell line, 6T3, restored regulated secretion of transfected POMC and the presence of immunoreactive CgA at the tips of the processes of these cells. Unlike CgA, CgB, another granin protein, could not substitute for the role of CgA in regulating LDCG biogenesis. Thus, we conclude that CgA is a key player in the regulation of the biogenesis of LDCGs in neuroendocrine cells. To examine the mechanism of sorting POMC to the LDCGs, we carried out site-directed mutagenesis, transfected the POMC mutants into PC12 cells, and assayed for regulated secretion. Our previous molecular modeling studies predicted a three-dimensional sorting motif in POMC that can bind to a sorting receptor, membrane carboxypeptidase E (CPE). The sorting signal consists of four conserved residues at the N-terminal loop structure of POMC: two acidic residues and two hydrophobic residues. The two acidic residues were predicted to bind to a domain on CPE (CPE254-273) containing two basic residues (R255 and K260) to effect sorting into immature secretory granules. Site-directed mutagenesis of the motif on POMC resulted in accumulation of the mutant in the Golgi, as well as high basal secretion, indicating that the mutant POMC was inefficiently sorted to the RSP. These results support the model that POMC is actively sorted to the RSP granules for processing and secretion by a sorting signal-mediated mechanism.