It has been demonstrated recently by acridine orange fluorescence that pancreatic zymogen granules are acidic in situ, with respect to the cytoplasm. To evaluate the relationship between the acidic intragranular pH and hormone-stimulated secretion, mouse pancreatic acini were treated with lysosomotropic agents to collapse the zymogen granule pH gradient. Methylamine, monensin, and chloroquine collapsed the granule pH gradient as evidenced by a disappearance of acridine orange fluorescence. Cholecystokinin octapeptide (CCK-8)-stimulated acinar amylase secretion was unaffected in the presence of up to 30 mM methylamine and slightly enhanced in the presence of 0.3-10 microM monensin or 3-300 microM chloroquine. Acini were also preincubated for 15 min before addition of either CCK-8 or bombesin with concentrations of the lysosomotropic agents that dissipated the granule acridine orange fluorescence within this time. With preincubation, basal amylase release was unaffected, while stimulated secretion was slightly enhanced by all three lysosomotropic agents. Monensin and methylamine caused vacuolization of Golgi and lysosomal membranes and inhibition of intracellular transport of newly synthesized proteins. Chloroquine affected lysosomes similarly but had little effect on Golgi membranes or on intracellular protein transport. We also demonstrate that parotid secretory granules are acidic in situ by the acridine orange technique. Thus acidified secretory granules may be a general feature of exocrine secretory granules, but the acid pH is not requisite for the final steps in protein secretion from isolated pancreatic acini.