We have characterized the regulation of expressed human ecto-ATPase (E-NTPDase 2), a cell surface integral membrane glycoprotein. Ecto-ATPase activity is inhibited by parameters that decrease membrane protein interaction, i.e., detergents and high temperatures. These inhibitory effects are overcome when membranes are pretreated with concanavalin A or chemical cross-linking agents that increase the amounts of ecto-ATPase oligomers. Cross-linking agents also abrogate substrate inactivation of the ecto-ATPase, a unique characteristic of the enzyme. These effects indicate that the magnitude of negative substrate regulation is dependent on quaternary structures of the protein, which likely involves interaction of transmembrane domains. The importance of transmembrane domains of ecto-ATPase in activity modulation is demonstrated further by the stimulatory effect of digitonin, a steroid glycoside that preferentially interacts with cholesterol in the membranes but does not promote oligomer formation. These results indicate that ecto-ATPase activity is regulated by a multitude of mechanisms, some of which may have physiological significance. Ecto-ATPase is also susceptible to transcriptional regulation. Ecto-ATPase gene expression is increased in a human hepatoma whereas it is undetectable in the normal liver.