Previously, we have shown that pancreatic acini release adenosine triphosphate (ATP) and ATP-handling enzymes, and pancreatic ducts express various purinergic P2 receptors. The aim of the present study was to establish whether pancreatic ducts also express adenosine receptors and whether these could be involved in secretory processes, which involve cystic fibrosis transmembrane regulator (CFTR) Cl- channels or Ca2+-activated Cl- channels and H(+)/HCO(-)(3) transporters. Reverse transcriptase polymerase chain reaction analysis on rat pancreatic ducts and human duct cell adenocarcinoma lines showed that they express A1, A2A, A2B, and A3 receptors. Real-time PCR revealed relatively low messenger RNA levels of adenosine receptors compared to beta-actin; the rank order for the receptors was A2A>A2B>or=A3>>A1 for rat pancreas and A2B>A2A>>A3>or=A1 for duct cell lines. Whole-cell patch-clamp recordings on rat pancreatic ducts showed that, in about half of the recordings, adenosine depolarized the membrane voltage, and this was because of the opening of Cl- channels. Using a Cl--sensitive fluorophore and single-cell imaging on duct cell lines, it was found that 58% of PANC-1 cells responded to adenosine, whereas only 9% of CFPAC-1 cells responded. Adenosine elicited Ca2+ signals only in a few rat and human duct cells, which did not seem to correlate with Cl- signals. A2A receptors were localized in the luminal membranes of rat pancreatic ducts, plasma membrane of many PANC-1 cells, but only a few CFPAC-1 cells. Taken together, our data indicate that A2A receptors open Cl- channels in pancreatic ducts cells with functional CFTR. We propose that adenosine can stimulate pancreatic secretion and, thereby, is an active player in the acini-to-duct signaling.