Alloantigen-specific and OVA-specific CD8+ CTL were shown here to express an ectoATPase. These CTL also express an ectoADPase, but do not express detectable levels of an ectoAMPase. CD8+ CTL transported adenosine into their cytoplasm at a rate of 2.3 x 10-11 mmol/min/105 cells. In contrast, adenosine uptake was 34-fold lower when ATP was used as the source of the nucleoside. This was consistent with the lack of ectoAMPase and suggests that the role of ectoATPase is not in the salvage of extracellular nucleotides. 5'-p-(fluorosulfonyl)benzoyl adenosine (5'-FSBA) is an ATP analogue affinity label that irreversibly inhibits CTL ectoATPase. Cells made ectoATPase activity deficient by modification with 5'-FSBA were not susceptible to potential lyric effects of extracellular ATP with less than 20% specific lysis at 20 mM of exogenous ATP. However, cells modified by 5'-FSBA were unable to kill their respective target cells. Complete inhibition of cell- mediated killing was observed with 1 mM 5'-FSBA. CTL modified by 5'-FSBA also failed to secrete TNF-α and IFN-γ after activation by the appropriate Ag. Killing was also inhibited by 5'-adenylylimidodiphosphate (a nonhydrolyzable ATP analogue), but not by ATP, ADP, α, β-methylene ADP (a nonhydrolyzable ADP analogue), AMP, or adenosine. Blockage of CTL activity by 5'-FSBA was not reversed by addition of ADP, suggesting that hydrolysis of ATP is an essential ectoATPase-mediated signal for CTL activation. These results suggest that ectoATPase is essential for Ag recognition and/or effector activities of CTL.