1 Transient accumulation of extracellular ATP reflects both release of ATP from intracellular stores and altered rates of ATP metabolism by ecto-enzymes. Ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) and ecto-nucleotide pyrophosphatases (eNPPs) degrade ATP, while ecto-nucleotide diphosphokinases (eNDPKs) synthesize ATP from ambient ADP. 2 Although the methylene ATP analogs betagamma-meATP and alphabeta-meATP are widely used as metabolically stable tools for the analysis of purinergic signaling, their specific effects on eNTPDase, eNPP, and eNDPK activities have not been defined. This study compared the actions of these analogs on extracellular ATP metabolism by human 1321N1 astrocytes, rat PC12 pheochomocytoma cells, and rat C6 glioma cells. 3 Both analogs significantly reduced clearance of extracellular ATP by 1321N1 cells that express both eNTPDases and eNPPs, as well as by C6 cells that exclusively express eNPPs. In contrast, both analogs were much less efficacious in inhibiting ATP clearance by PC12 cells that predominantly express eNTPDases. Betagamma-meATP, but not alphabeta-meATP, was effectively hydrolyzed by the 1321N1 and C6 cells; PC12 cells did not significantly degrade this analog. 4 Alphabeta-meATP, but not betagamma-meATP, acted as a substrate for purified yeast NDPK to generate ATP via trans-phosphorylation of ADP. alphabeta-meATP also acted as substrate for the eNDPK activities expressed by 1321N1, PC12, and C6 cells and thereby induced extracellular ATP accumulation in the presence of ambient or exogenously added ADP. 5 These results indicate that methylene ATP analogs exert complex and cell-specific effects on extracellular ATP metabolism that can significantly modify interpretation of studies that use these reagents as probes of purinergic signal transduction in intact tissues.