Pancreatic duct epithelial cells (PDEC) mediate the secretion of fluid and electrolytes and are exposed to refluxed bile. In nontransformed cultured dog PDEC, which express many ion transport pathways of PDEC, 1 mM taurodeoxycholic acid (TDCA) stimulated an ¹²⁵I efflux inhibited by DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and a ⁸⁶Rb⁺ efflux inhibited by charybdotoxin. Inhibition by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suggests mediation via increased intracellular Ca²⁺ concentration, whereas the absence of lactate dehydrogenase release excludes cellular toxicity. At 1 mM, TDCA stimulated a larger ¹²⁵I efflux than glycodeoxycholate; two dihydroxy bile acids, taurochenodeoxycholate and TDCA, were similarly effective, whereas a trihydroxy bile acid, taurocholate, was ineffective. In Ussing chambers, 1 mM serosal or 2 mM luminal TDCA stimulated an I_sc increase from confluent PDEC monolayers. TDCA also stimulated 1) a short-circuit current (I_sc) increase from basolaterally permeabilized PDEC subject to a serosal-to-luminal Cl gradient that was inhibited by BAPTA-AM, DIDS, and NPPB and 2) an I_sc increase from apically permeabilized PDEC subject to a luminal-to-serosal K⁺ gradient inhibited by BAPTA-AM and charybdotoxin. Along with the efflux studies, these findings suggest that TDCA interacts directly with PDEC to stimulate Ca²⁺-activated apical Cl channels and basolateral K⁺ channels. Monolayer transepithelial resistance was only minimally affected by 1 mM serosal and 2 mM luminal TDCA but decreased after exposure to higher TDCA concentrations (2 mM serosal and 4 mM luminal). A secretory role for bile acids should be considered in pancreatic diseases associated with bile reflux.