The vacuolar H(+)-ATPase (V-ATPase) establishes pH gradients along secretory and endocytic pathways. Progressive acidification is essential for proteolytic processing of prohormones and aggregation of soluble content proteins. The V-ATPase V(0) subunit is thought to have a separate role in budding and fusion events. Prolonged treatment of professional secretory cells with selective V-ATPase inhibitors (bafilomycin A1, concanamycin A) was used to investigate its role in secretory-granule biogenesis. As expected, these inhibitors eliminated regulated secretion and blocked prohormone processing. Drug treatment caused the formation of large, mixed organelles, with components of immature granules and lysosomes and some markers of autophagy. Markers of the trans-Golgi network and earlier secretory pathway were unaffected. Ammonium chloride and methylamine treatment blocked acidification to a similar extent as the V-ATPase inhibitors without producing mixed organelles. Newly synthesized granule content proteins appeared in mixed organelles, whereas mature secretory granules were spared. Following concanamycin treatment, selected membrane proteins enter tubulovesicular structures budding into the interior of mixed organelles. shRNA-mediated knockdown of the proteolipid subunit of V(0) also caused vesiculation of immature granules. Thus, V-ATPase has a role in protein sorting in immature granules that is distinct from its role in acidification.