ATP is a signaling molecule that initiates various cellular responses after binding to P2X and P2Y receptors. These two classes of receptors are expressed in the pancreatic duct cells where they regulate ion and fluid transport (1). Our previous studies have shown that ATP is secreted from acinar zymogen granules into duct lumen (2). The aim of the present studies was to establish whether ATP is also released locally by pancreatic ducts and to determine which physiological and pathophysiological stimuli have such effects, and possible involvement of VNUT (Vesicular Nucleotide Transporter). Luciferase/luciferin bioluminescence assay was used to determine concentrations of ATP released from human pancreatic duct cells (e.g. Capan-1) in response to various stimulants and Fluo4-AM was used for detecting calcium changes. Results are given as mean changes from basal values ± S.E.M; p<0.05 accepted as significant. We also applied PCR and Western blot to investigate VNUT expression. We observed fast and continuous ATP release in Capan-1 cells in response to hypotonic and mechanical stimuli of 2.0±1.4 nM/106 cells/ml (n=6) and 0.9±0.5 µM/106 cells/ml (n=7). Ionomycin (5 µM) also induced similar ATP release 1.6 ± 0.9 nM/106 cells/ml (n=6). Ethanol (10-100 mM) had no effect on extracellular ATP (n=5). Interestingly, agents that act via receptors induced significant ATP release. For example, addition of UTP (1-10 µM) caused ATP release of 3.2 ± 0.7 nM/106 cells/ml (n=7). The bile acid chenodeoxycholate (CDC) induced dose-dependent (0.1-1 mM), fast and maximal ATP release of 1.0±0.3 µM/106 cells/ml (n=5). Concomitantly, CDC induced increase in cellular Ca2+ and a decrease in intracellular ATP, as detected by fluorescent markers (Fluo-4; Mg-Green) and confocal microscopy (n=4). Fluorescent ATP store markers (quinacrine, MANT-ATP), use of agents that interfere with secretory pathways (n=4) and molecular evidence for VNUT (n=5), indicated that ducts cells contain vesicular ATP stores. Together, our data show that there are various stimuli causing ATP release with different kinetics and this may indicate different releasing mechanisms, including vesicular release. In pancreatic ducts, ATP release might be an important co-stimulatory and protective mechanism for mechanical stimuli, as well as for substances that can enter lumen of pancreatic ducts, e.g. CDC and UTP. In low concentrations ATP can stimulate opening of Cl- and K+ channels and fluid secretion (3). However, overstimulation depletes the cells of ATP, partially via ATP release, and can therefore be involved in pathological changes in pancreas.