The presence of one or more calcium-dependent ecto-ATPases (enzymes that hydrolyze extracellular 5'-triphosphates) in mammalian taste buds was first shown histochemically. Recent studies have established that dominant ecto-ATPases consist of enzymes now called nucleoside triphosphate diphosphohydrolases (NTPDases). Massively parallel signature sequencing (MPSS) from murine taste epithelium provided molecular evidence suggesting that NTPDase2 is the most likely member present in mouse taste papillae. Immunocytochemical and enzyme histochemical staining verified the presence of NTPDase2 associated with plasma membranes in a large number of cells within all mouse taste buds. To determine which of the three taste cell types expresses this enzyme, double-label assays were performed with antisera directed against the glial glutamate/aspartate transporter (GLAST), the transduction pathway proteins phospholipase Cbeta2 (PLCbeta2) or the G-protein subunit alpha-gustducin, and serotonin (5HT) as markers of type I, II, and III taste cells, respectively. Analysis of the double-labeled sections indicates that NTPDase2 immunoreactivity is found on cell processes that often envelop other taste cells, reminiscent of type I cells. In agreement with this observation, NTPDase2 was located to the same membrane as GLAST, indicating that this enzyme is present in type I cells. The presence of ecto-ATPase in taste buds likely reflects the importance of ATP as an intercellular signaling molecule in this system.