Adenosine modulates different functional activities in many cells of the gastrointestinal tract; some of them are believed to be mediated by interaction with its four G-protein-coupled receptors. The renewed interest in the adenosine A_2B receptor (A2BR) subtype can be traced by studies in which the introduction of new genetic and chemical tools has widened the pharmacological and structural knowledge of this receptor as well as its potential therapeutic use in cancer and inflammation- or hypoxia-related pathologies. In the acid-secreting parietal cells of the gastric mucosa, the use of various radioligands for adenosine receptors suggested the presence of the A₂ adenosine receptor subtype(s) on the cell surface. Recently, we confirmed A2BR expression in native, non-transformed parietal cells at rest by using flow cytometry and confocal microscopy. In this study, we show that A2BR is functional in primary rabbit gastric parietal cells, as indicated by the fact that agonist binding to A2BR increased adenylate cyclase activity and acid production. In addition, both acid production and radioligand binding of adenosine analogues to isolated cell membranes were potently blocked by selective A2BR antagonists, whereas ligands for A₁, A_2A and A₃ adenosine receptors failed to abolish activation. We conclude that rabbit gastric parietal cells possess functional A2BR proteins that are coupled to Gs and stimulate HCL production upon activation. Whether adenosine- and A2BR-mediated functional responses play a role in human gastric pathophysiology is yet to be elucidated.