T-cell activation requires the influx of extracellular calcium, although mechanistic details regarding such activation are not fully defined. Here, we show that P2X 7 receptors play a key role in calcium influx and downstream signaling events associated with the activation of T cells. By real-time PCR and immunohistochemistry, we find that Jurkat T cells and human CD4+ T cells express abundant P2X 7 receptors. We show, using a novel fluorescent microscopy technique, that T-cell receptor (TCR) stimulation triggers the rapid release of ATP (< 100 μM). This release of ATP is required for TCR-mediated calcium influx, NFAT activation, and interleukin-2 (IL-2) production. TCR activation up-regulates P2X 7 receptor gene expression. Removal of extracellular ATP by apyrase or alkaline phosphatase treatment, inhibition of ATP release with the maxi-anion channel blocker gadolinium chloride, or siRNA silencing of P2X 7 receptors blocks calcium entry and inhibits T-cell activation. Moreover, lymphocyte activation is impaired in C57BL/6 mice that express poorly functional P2X 7 receptors, compared to control BALB/c mice, which express fully functional P2X 7 receptors. We conclude that ATP release and autocrine, positive feedback through P2X 7 receptors is required for the effective activation of T cells.—Yip, L., Woehrle, T., Corriden, R., Hirsh, M., Chen, Y., Inoue, Y., Ferrari, V., Insel, PA, Junger, WG Autocrine regulation of T-cell activation by ATP release and P2X 7 receptors.