An extracellular membrane-associated ectoATPase has been identified on the human natural killer cell line NK3.3. The enzyme is distinct from other classes of ATPases, kinases, and phosphatases. NK3.3 ectoATPase demonstrated a Km for ATP of 41 microM and a Vmax of 0.2 mumol/min and required both Ca2+ and Mg2+ for maximal activity. Purine and pyrimidine nucleotides were competitive inhibitors of the catalytic reaction. Inhibition increased with the addition of increasing negative charge of the phosphate side chain and was also dependent on contributions from the nucleoside. NK3.3 ectoATPase activity was inhibited by reaction with the affinity label [p-(fluorosulfonyl)benzoyl]-5'-adenosine (5'-FSBA), which is shown to modify the enzyme at or near the ATP-binding domain. Photoaffinity labeling of intact NK3.3 cells with [alpha-32P]-8-azidoATP demonstrated an ATP-binding protein of 68-80 kDa unique to NK3.3 cells. A positive correlation was observed between the ability of the various nucleotides to block photoincorporation into the 68-80-kDa protein and their ability to inhibit ectoATPase activity. NK3.3 cells which were made ectoATPase-deficient by reaction with 5'-FSBA demonstrated that this enzyme does not have a major role in the protection of this cytolytic effector cell from the possible lytic effects of extracellular ATP.