Two isoforms of ATP diphosphohydrolase (ATPDase; EC 3.6.1.5) have been previously characterized, purified, and identified. This enzyme is an ectonucleotidase that catalyzes the sequential release of gamma- and beta-phosphate groups of triphospho- and diphosphonucleosides. One of its putative roles is to modulate the extracellular concentrations of purines in different physiological systems. The purpose of this study was to define, identify, and localize these two isoforms of ATPDase in the pig digestive system. ATPDase activity was measured in pig stomach, duodenum, pancreas, and parotid gland. Enzyme assays, electrophoretograms, and Western blots with a polyclonal antibody that recognizes both isoforms demonstrate the presence of ATPDase in these organs. Immunolocalization showed intense reactions with gastric glands (parietal and chief cells), intestine (columnar epithelial cells), parotid gland, and pancreas. Smooth muscle cells all along the digestive tract were also highly reactive. Considering the variety of purinoceptors associated with the digestive system, the ATPDase is strategically positioned to modulate purine-mediated actions such as electrolyte secretion, glandular secretion, smooth muscle contraction, and blood flow.