Ecto-ATPase, a transmembrane enzyme that catalyzes the hydrolysis of extracellular ATP (ATP(e)) to ADP and inorganic phosphate, is expressed upon cell activation. Ecto-ATPase is inhibited by non-hydrolyzable ATP analogues, which are competitive inhibitors of the catalytic reaction, and the ATP analogue affinity label, 5'-p-(fluorosulfonyl)benzoyl adenosine (5'-FSBA), which irreversibly inhibits the catalytic activity. These nucleotide antagonists do not cross the cell membrane and are specific for ecto-ATPase in T cells, B cells and NK cells. Inhibition of ecto-ATPase by both reversible and irreversible nucleotide antagonists results in the inhibition of antigen-induced cytokine secretion and cytolytic activity of T cells. Likewise, granule release and cytolytic activity of NK cells as well as antibody secretion and spontaneous proliferation by B-cell hybridomas are inhibited. Inhibition of ecto-ATPase does not influence effector cell-target cell conjugate formation, but acts, in part, by regulating the influx of extracellular calcium that is necessary to maintain cellular activation. Thus, further elucidation of ecto-ATPase regulation and expression and its interaction with intracellular signal transduction events will provide a basis for understanding the role of the hydrolysis of ATP(e) by ecto-ATPase in lymphocyte effector function.