Urinary tract infections are commonly caused by α-hemolysin (HlyA)-producing Escherichia coli. In erythrocytes, the cytotoxic effect of HlyA is strongly amplified by P2X receptors, which are activated by extracellular ATP released from the cytosol of the erythrocytes. In renal epithelia, HlyA causes reversible [Ca(2+)]i oscillations, which trigger interleukin-6 (IL-6) and IL-8 release. We speculate that this effect is caused by HlyA-induced ATP release from the epithelial cells and successive P2 receptor activation. Here, we demonstrate that HlyA-induced [Ca(2+)]i oscillations in renal epithelia were completely prevented by scavenging extracellular ATP. In accordance, HlyA was unable to inflict any [Ca(2+)]i oscillations in 132-1N1 cells, which lack P2R completely. After transfecting these cells with the hP2Y2 receptor, HlyA readily triggered [Ca(2+)]i oscillations, which were abolished by P2 receptor antagonists. Moreover, HlyA-induced [Ca(2+)]i oscillations were markedly reduced in medullary thick ascending limbs isolated from P2Y2 receptor-deficient mice compared with wild type. Interestingly, the following HlyA-induced IL-6 release was absent in P2Y2 receptor-deficient mice. This suggests that HlyA induces ATP release from renal epithelia, which via P2Y2 receptors is the main mediator of HlyA-induced [Ca(2+)]i oscillations and IL-6 release. This supports the notion that ATP signaling occurs early during bacterial infection and is a key player in the further inflammatory response.