P2X₇ receptors are nonselective cation channels opened by the binding of extracellular ATP^4-. They are expressed in the cell membrane, but also in intracellular vesicles of cells of the immune system. They are involved in the modulation of the immune system, inflammation and nociception. Here, we investigated the effect of extracellular anions on P2X₇ receptor channel gating and permeation. Human P2X₇ receptors were expressed in Xenopus laevis oocytes. Cell membrane currents were measured by the two microelectrode voltage clamp technique. If extracellular Cl^- was substituted by organic anions like glutamate or aspartate, the ATP-induced P2X₇ receptor-mediated currents were increased. In contrast, if Cl^- was replaced by inorganic anions like nitrate, sulfate or iodide, the P2X₇ receptor-dependent currents were inhibited. We measured ATP concentration-response curves in bathing solutions containing either Cl^-, glutamate or iodide as extracellular anions replacing Cl^-. The main Cl^- substitution effect was that glutamate increased and iodide decreased the agonist efficacy of high ATP concentrations. However, at low ATP concentrations, Cl^- substitution by glutamate or iodide was without effect on the P2X₇ receptor activation. For further analysis of the anion effect on P2X₇ receptors, we performed single channel current measurements by the patch clamp technique in the outside-out configuration. While Cl^- substitution did not affect the single channel conductance, the P2X₇ receptor channel open probability was increased or decreased if Cl^- was replaced by glutamate or iodide, respectively. We conclude that anions affect ion channel opening steps after binding of all three ATP^4- molecules on the P2X₇ receptor.