Previous studies of enzyme secretion from isolated pancreatic acinar cells and of isolated zymogen granules (ZG) have reported that both a Cl- and a K+ permeability are present on the ZG membrane. It has been suggested that ion influx via these permeability pathways, followed by water movement is required for granular swelling which appears to be intimately related to exocytosis. However, little is known about the regulation of these pathways by secretagogues. Evidence suggests that cAMP-protein kinase A and diacylglycerol-protein kinase C are second messengers in stimulation of exocytosis. In the present study we have examined ion permeability pathways in ZG isolated from control cells and from cells pretreated with the acetylcholine analog carbachol (Cch), with the peptide hormone cholecystokinin (CCK) and with second messengers of hormone action such as cAMP and the diacylglycerol analog 12-O-tetradecanoyl phorbol-13-acetate (TPA). Ion and water influx rates in ZG and consequent swelling and lysis of granules was monitored by measuring changes in optical densities of ZG suspensions at 540 nm following additions of the electrogenic or electroneutral ionophores valinomycin and nigericin, respectively. The data show that both a Cl- conductance and an anion exchange pathway are present in the granule membrane. Both pathways are activated by pretreatment of isolated cells with CCK or of isolated permeabilised cells with cAMP, whereas only the Cl- conductance is increased by pretreatment with Cch or with TPA. The anion transport inhibitor DIDS abolishes Cl- conductance but has no effect on the anion exchanger. Granular lysis is also inhibited by buffers of high osmotic strength. The direct application of the catalytic subunit of protein kinase A (PKA) and ATP to isolated ZG inhibits both the Cl- conductance and the anion exchange pathway probably mediated by phosphorylation. This result does not seem to be consistent with the observation that in isolated ZG from cAMP prestimulated cells both Cl- conductance and the Cl-/anion exchanger are activated. We conclude that stimulation of pancreatic acinar cells by secretagogues results in activation of Cl- permeability pathways in the ZG membrane mediated by cAMP-protein kinase A and DG-protein kinase C mediated phosphorylation. It is possible that in addition to "on" reactions also "off" reactions exist which close the Cl- channel, e.g. by dephosphorylation and that in activation of Cl- permeability pathways different sites for cAMP-protein kinase A action or different protein kinases A are involved. © 1989 Springer-Verlag.