The membrane permeability of pancreatic zymogen granules was evaluated in vitro with granules isolated from rats in different secretory states: 1) untreated, 2) pretreated with a muscarinic antagonist, 3) pretreated with a muscarinic and an adrenergic antagonist, 4) pretreated as in 3 and then stimulated with the secretagogue cholecystokinin 4 min before death, and 5) pretreated as in 3 and then stimulated with the secretagogue secretin 4 min before death. Granules isolated from untreated rats had variable ionic permeabilities but in general possessed both chloride conductance and electroneutral exchange pathways with low permeabilities to alkali metal ions. In contrast, granules from animals pretreated with secretory antagonists had very low ion permeabilities to both inorganic anions, such as chloride, and alkali metal ions. Injection of the peptide secretagogues cholecystokinin or secretin resulted in a relatively fast (within 4 min) activation or induction of high chloride permeabilities through both chloride conductance and chloride/hydroxide (or chloride/bicarbonate) exchange pathways. In addition, the secretagogues increased the cation permeability of the granule membrane, which exhibited a distinct potassium selectivity. Chloride conductance has been postulated to play a major role in fluid secretion coupled to exocytosis of macromolecules [R. C. DeLisle and U. Hopfer, Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G489-G496, 1986]. These results demonstrate that granules may actively participate in the secretory process and suggest that some of the physiological targets in the cascade of events leading to secretion are anion and cation transporters in the zymogen granule membrane.