Extracellular ATP and other nucleotides transmit signals to cells via surface-associated molecules whose binding sites face the extracellular milieu. Ecto-nucleoside triphosphate diphosphohydrolase is such an ATP-binding enzyme that is expressed by activated lymphocytes. We have previously shown that nonhydrolyzable ATP analogs block the lytic activity of NK cells and CD8+ T cells as well as their E-NTPDase activity. These results suggest that the hydrolysis of ATP may play a role in lymphocyte function. Here we report that E-NTPDase activity is up-regulated within 15 min of T cell stimulation and that reversible and irreversible enzyme inhibitors profoundly reduce secretion of IL-2 and IFN-γ, but not IL-4. TNF-α, IL-10, and IL-5 production showed intermediate sensitivity to these ATP analogs. Depletion of extracellular ATP also inhibited secretion of IFN-γ, but not IL-4, supporting the interpretation that extracellular ATP is required for secretion of some, but not all, cytokines. E-NTPDase antagonists reduced transcription of IL-2 mRNA and inhibited TCR-mediated intracellular calcium flux. These results suggest that extracellular ATP plays an essential role in the TCR-mediated signal transduction cascade for expression of certain cytokine genes.