Introduction. We have recently shown that a high dose (1mM) of the non-conjugated bile acid chenodeoxycholate (CDC) had strong inhibitory effects on the activities of acid/base transporters on pancreatic ductal epithelial cells (PDEC) (Ref 1). However, the same dose of conjugated glycochenodeoxycholate (GCDC) had no effect on the ion transporters. In addition, a high dose of CDC evoked a toxic sustained calcium elevation. Our aim was to characterize the intracellular mechanisms by which the ion transport mechanisms are inhibited. Methods. Intra/interlobular pancreatic ducts were isolated from the pancreas of guinea pigs. High dose (1mM) of CDC or GCDC was administered basolaterally for 1-10 minutes. Intracellular pH (pH_i) of PDEC were measured by microfluorometry using BCECF. The intracellular ATP level (ATP)_i was determined using Mg-Green which has been shown to indirectly reflect the changes in (ATP)_i (Ref 2). Morphological changes of PDEC were evaluated by transmission electron microscopy. Results. Administration of a low dose (0.1mM) of CDC or GCDC for 10 min had no effects on the intracellular organelles. In addition, a high dose (1mM) of the conjugated GCDC did not induce morphological changes. Importantly, exposure of 1mM CDC for 10 min strongly damaged all of the mitochondria. The mitochondria swelled up and their inner membranes were disrupted. Other intracellular organelles such as nuclei or Golgi apparatus seemed to be unaltered. For positive control experiments we used the mitochondrial toxin carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 100μM) which totally mimicked the effect of CDC on mitochondria. Next we set out to investigate whether (ATP)_i is affected due to the mitochondrial damage. Administration of a low dose of CDC or GCDC for 10 min had no effect on the (ATP)_i, however, a high dose of CDC and CCCP markedly and irreversibly reduced the (ATP)_i. Although 1mM GCDC also decreased (ATP)_i, this depletion was reversible and significantly less than the depletion caused by CDC or CCCP. Finally, we provided evidence that (ATP)_i depletion is crucial in the toxic inhibitory effect of CDC on the ion transporters. To characterize the effects of (ATP)_i depletion on the activities of NHE, NBC and CBE, we used the NH₄Cl pulse technique in HCO₃^–-buffered solution. CCCP strongly inhibited NBC, NHE (recovery from acid load) and CBE (recovery from alkali load). Conclusions. In conclusion, these results clearly show that (i) a high dose of the non-conjugated CDC, but not the conjugated GCDC, causes mitochondrial damage followed by (ATP)_i depletion and (ii) the (ATP)_i depletion by itself can be responsible for the impaired fluid and HCO₃^– secretion.