There is growing evidence that P2X₇ receptors (P2X₇R) couple to rapid activation of NADPH oxidase leading to the generation of reactive oxygen or nitrogen species (RONS) (Parvathenani et al, 2003; Suh et al, 2001; Ferrari et al, 2000). In this study, we have investigated the action of extracellular adenosine 5′ triphosphate (ATP) on the oxidative state of differentiated THP-1 macrophages loaded with the RONS-sensitive fluorescent dye dichlorofluorescin diacetate. All experiments were performed in a physiological salt saline containing (in mM) 130 NaCl, 5 KCl, 1.5 CaCl₂, 1 MgCl₂, 5 NaHCO₃, 1.5 KH₂PO₄, 25 HEPES, 10 glucose (pH 7.3 with NaOH). All data expressed as mean ± s.e.m. with number of experiments in parenthesis and statistical analysis was performed using Student’s T-test. Stimulation of differentiated THP-1 macrophages with extracellular ATP triggered a dose dependent burst of RONS formation within 30 s (ATP EC50 1.96 ± 0.26 mM (n = 3)) that was inhibited using the P2X₇R inhibitor 1-[N,O-bis-(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62; IC50 368.1 ± 273.1 nM, n = 3). The addition of extracellular superoxide dismutase (≥10 U/ml, n = 4) completely blocked RONS formation suggesting that the RONS sensitive dye was measuring peroxynitrite generation and not hydrogen peroxide. ATP-induced RONS generation was inhibited using N-acetylcysteine (an antioxidant, IC50 86 ± 5.5 µM, n = 3) or diphenyleneiodonium (IC50 11.7 ± 5.6 µM, n = 3), partially inhibited with the inducible nitiric oxide synthase (iNOS) inhibitor 1400W (1 µM, 77.9 % control, n = 2, p = 0.024), but not with allopurinol. Therefore indicating NADPH oxidase and iNOS are responsible for the generation of RONS. Finally, bromoenol lactone (BEL) inhibits calcium-independent phospholipase A2 (iPLA2); incubation of cells with BEL inhibited ATP-mediated RONS generation (IC50 3.7 ± 1.5 µM, n = 3). Co-application of exogenous arachidonic acid (10 µM) with 3 mM ATP was able to recover BEL (10 µM)-inhibited RONS generation suggesting the involvement of iPLA2 in the intracellular signalling process from P2X₇R to RONS generation. To conclude, these results demonstrate that human macrophage P2X₇Rs rapidly engage NADPH oxidase triggering the generation of peroxynitrite. This pathway may play an important role in host defence and inflammation.