Ca2+-induced enzyme secretion in the exocrine pancreas is not completely understood. We have proposed that Ca2+-induced enzyme secretion in the exocrine pancreas involves activation of ion conductances in the membrane of zymogen granules (ZG). Here we have identified a Ca 2+-activated anion conductance in rat pancreatic ZG membranes (ZGM). Ca2+ (2.5-50μM) increased the conductance for I-, NO3-, Br-, or HCO3-, but not for Cl-, as determined by the rate of valinomycin-induced osmotic lysis of ZG suspended in isotonic K+-salts. 4,4 ′-Diisothiocyanatodihydrostilbene-2,2′- disulfonate (100μM) or 25μM dithiothreitol strongly inhibited Ca 2+-dependent lysis. The permeability sequence, Ca2+ dependence, and inhibitor sensitivity of ZG anion conductance are reminiscent of a family of epithelial Ca2+-activated anion channels (CLCA). CLCA expression was confirmed by RT-PCR with rat pancreatic mRNA and mouse CLCA1 primers. A PCR product (580bp) exhibited 81%, 77%, and 57% amino acid similarity to the three mouse isoforms mCLCA-1, -2, and -3 (mgob-5), respectively. Antibodies against bovine tracheal CLCA1 showed CLCA expression in ZGM by immunoblotting, immunoperoxidase light microscopy, and immunogold labeling. These findings suggest that a CLCA-related protein could account for the Ca2+-activated HCO3- conductance of rat pancreatic ZGM and contribute to hormone-stimulated enzyme secretion. © 2002 Elsevier Science (USA). All rights reserved.