Pancreatic duct epithelial cells (PDECs) mediate the pancreatic secretion of fluid and electrolytes. Membrane K+ channels on these cells regulate intracellular K+ concentration; in combination with the Na+/H+ antiport and Na+, K+-adenosine triphosphatase (ATPase), they may also mediate serosal H+ secretion, balancing luminal HCO− 3 secretion. We describe the K+ conductances on well-differentiated and functional nontransformed cultured dog PDECs. Through 86 Rb+ efflux studies, we demonstrated Ca 2+-activated K+ channels that were stimulated by A23187, thapsigargin, and 1-ethyl-2-benzimidazolinone, but not forskolin. These conductances also were localized on the basolateral membrane because 86 Rb+ efflux was directed toward the serosal compartment. Of the K+ channel blockers, BaCl 2, charybdotoxin, clotrimazole, and quinidine, but not 4-aminopyridine, apamin, tetraethylammonium, or iberiotoxin, inhibited 86 Rb+ efflux. This efflux was not inhibited by amiloride, ouabain, and bumetanide, inhibitors of the Na+/H+ antiport, the Na+, K+-ATPase pump, and the Na+, K+, 2Cl− cotransporter, respectively. When apically permeabilized PDEC monolayers were mounted in Ussing chambers with a luminal-to-serosal K+ gradient, A23187 and 1-ethyl-2-benzimidazolinone stimulated a charybdotoxin-sensitive shortcircuit current (I sc) increase. Characterization of K+ channels on these cultured PDECs, along with previous identification of Cl− channels (1), further supports the importance of these cells as models for pancreatic duct secretion.