Two tyrosine sulfated proteins, chromogranin B and secretogranin II, are targeted into regulated secretory granules, which are stored until stimulation causes them to fuse with the plasma membrane. In PC12 cells, [35S]-sulfate labels chromogranin B, secretogranin II and proteoglycans just before they are sorted into the regulated and constitutive secretory pathways. Chromogranin B was secreted by both the regulated and constitutive secretory pathways. In contrast, very little secretogranin II was secreted constitutively. Formation of regulated and constitutive secretory vesicles from the trans Golgi network was reconstituted in vitro. Mechanically permeabilized PC12 cells released regulated and constitutive vesicles containing sulfated markers. At early chase times, chromogranin B was predominantly released from permeabilized cells in small vesicles that comigrated with constitutive secretory vesicles containing proteoglycans. At intermediate chase times the vesicles containing chromogranin B resembled immature secretory granules containing secretogranin II. At very long chase times chromogranin B and secretogranin II were found in mature secretory granules that did not escape the permeabilized cells in vitro. We conclude that chromogranin B is sorted into immature regulated secretory granules with lower efficiency than secretogranin II.