Integral membrane proteins of neuroendocine dense-core vesicles (DCV) appear to undergo multiple rounds of exocytosis; however, their trafficking and site of incorporation into nascent DCVs is unclear. Previous studies with phogrin (IA-2beta) identified sorting signals in the luminal domain that is cleaved post-translationally; we now describe an independent DCV targeting motif in the cytosolic domain that may function at the level of endocytosis and recycling. Pulse-chase radiolabeling and cell surface biotinylation experiments in the pituitary corticotroph cell line AtT20 showed that the mature 60/65 kDa form that resides in the DCV is generated by limited proteolysis in a post-trans Golgi network compartment with similar kinetics to the formation of the principal cargo, ACTH. Phogrin is exposed on the cell surface in response to stimuli and progressively internalized to a perinuclear compartment that overlaps with recycling endosomes marked by transferrin. Chimeric molecules of phogrin transmembrane and cytosolic sequences with the interleukin-2 receptor alpha chain (Tac) were sorted to DCVs through the action of an extended tyrosine-based motif Y(654)QELCRQRMA located in a 27aa sequence adjacent to the membrane-spanning domain. A 36aa domain terminating in this sequence conferred DCV localization to Tac in the absence of any other cytosolic or luminal phogrin components. The endocytosis and DCV targeting of phogrin Y(654) > A mutants correlated with the impaired binding of the phogrin cytosolic tail to the micro-subunit of the AP2 adaptor complex in vitro.