Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, endocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway is unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (GFP), or chemiluminescent (EAP) reporters, to identify trafficking determinants mediating DCG-targeting of SgII in sympathoadrenal cells. 3-D deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs, and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/TGN. Truncation analyses reveal the presence of DCG-targeting signals within both N-terminal and C-terminal regions of SgII, with the putative a-helix-containing SgII25-41 and SgII334-348 acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells, and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure, and/or interaction with a specific component of the DCG membrane.