Endocrine and neuronal cells have highly developed secretion mechanisms, and the secretion can be either constitutive or regulated by physiological stimuli. In the constitutive pathway, intracellular transport vesicles undergo immediate fusion reactions after arrival at the target. In regulated secretion, vesicles accumulate near the target membrane until triggered to fuse, typically by a local rise in free Ca 2ϩ . In the present study, we characterize the processing and secretion mechanisms of the glial cell line-derived neurotrophic factor (GDNF). Although the function of GDNF has been extensively studied, very little is known about the basic cell biology of GDNF and its precursor forms (␣)pro-GDNF and (␤)pro-GDNF that have different pro-regions. Our results show that both (␣)pro-GDNF and (␤)pro-GDNF are secreted. We demonstrate that KCl-induced depolarization increases the secretion of (␤)pro-GDNF and corresponding mature GDNF, but not (␣)pro-GDNF and corresponding mature GDNF, to the cell medium in a Ca 2ϩ -dependent manner. In parallel with this, immunofluorescence analysis of cells show that (␣)pro-GDNF/GDNF is localized mostly in the Golgi complex, whereas (␤)pro-GDNF/GDNF is localized primarily in secretogranin II and Rab3A-positive vesicles of the regulated secretory pathway. In addition, we find that matrix metalloproteinases and plasmin that cleave pro-BDNF and pro-NGF are not responsible for the cleavage of pro-GDNF, whereas furin endoproteinase, PACE4, and proprotein convertases PC5A, PC5B, and PC7 can cleave pro-GDNF into mature GDNF. Thus, the processing and secretion mechanisms of GDNF are different from those of BDNF and NGF.