Adenosine regulates exocrine and endocrine secretions in the pancreas. Adenosine is considered to play a role in acini-to-duct signaling in the exocrine pancreas. To identify the molecular basis of functional adenosine receptors in the exocrine pancreas, immunohistochemical analysis was performed in the rat, mouse, and guinea pig pancreas, and the secretory rate and concentration of HCO3- in pancreatic juice from the rat pancreas were measured. The A2A adenosine receptor colocalized with ezrin, an A-kinase anchoring protein, in the luminal membrane of duct cells in the mouse and guinea pig pancreas. However, a strong signal ascribed to A2B adenosine receptors was detected in insulin-positive β cells in islets of Langerhans. The A2A adenosine receptor agonist 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS 21680) stimulated HCO3--rich fluid secretion from the rat pancreas. These results indicate that A2A adenosine receptors may be, at least in part, involved in the exocrine secretion of pancreatic duct cells via acini-to-duct signaling. The adenosine receptors may be a potential therapeutic target for cancer as well as exocrine dysfunctions of the pancreas.