Our laboratory has been using a permeabilized cell system derived from rat anterior pituitary GH3 cells expressing prosomatostatin (pro-SRIF) to study prohormone processing and nascent secretory vesicle formation in vitro. Because calcium is necessary for prohormone processing enzyme activity, secretory granule fusion with the plasma membrane, and possibly sorting to the regulated pathway, we treated permeabilized cells with the calcium ionophore A23187 to determine the role of calcium in pro-SRIF cleavage and nascent vesicle formation from the trans-Golgi network (TGN). Here we demonstrate that pro-SRIF cleavage was markedly inhibited when lumenal free calcium was chelated with EGTA in the presence of A23187. Surprisingly, submillimolar free calcium (approximately 15 microM) was sufficient to maintain prohormone cleavage efficiency, a value far lower than that estimated for total calcium levels in the TGN and secretory granules. Experiments using both A23187 and the protonophore CCCP revealed that free calcium is absolutely required for efficient pro-SRIF cleavage, even at the optimal pH of 6.1. Secretory vesicle formation by contrast was not inhibited by calcium chelation but rather by millimolar extralumenal free calcium. Together, these observations demonstrate that pro-SRIF processing and budding of nascent secretory vesicles from the TGN can be uncoupled and therefore have distinct biochemical requirements. Interestingly, our data using intact GH3 cells demonstrate that basal secretion of SRIF-related material is largely calcium-dependent and therefore cannot be equated with constitutive pathway secretion. These results underscore the importance of determining calcium requirements before assigning a secretion event to either the constitutive or regulated secretory pathway.