The presence of P2X7 on erythroid cells is well established, but its physiological role remains unclear. The current study aimed to determine if P2X7 activation induces reactive oxygen species (ROS) formation in murine erythroleukaemia (MEL) cells, a commonly used erythroid cell line. ATP induced ROS formation in a time- and concentration-dependent fashion. The most potent P2X7 agonist, 2′(3′)-O-(4-benzoylbenzoyl)ATP, but not UTP or ADP, also induced ROS formation. The P2X7 antagonist, A-438079, impaired ATP-induced ROS formation. The ROS scavenger, N-acetyl-l-cysteine, and the ROS inhibitor, diphenyleneiodonium, also impaired P2X7-induced ROS formation, but use of enzyme-specific ROS inhibitors failed to identify the intracellular source of P2X7-induced ROS formation. P2X7-induced ROS formation was impaired partly by physiological concentrations of Ca2+ and Mg2+ and almost completely in cells in N-methyl-d-glucamine chloride medium. The p38 MAPK inhibitors SB202190 and SB203580, and the caspase inhibitor Z-VAD-FMK, but not N-acetyl-l-cysteine, impaired P2X7-induced MEL cell apoptosis. ATP also stimulated p38 MAPK and caspase activation, both of which could be impaired by A-438079. In conclusion, these findings indicate that P2X7 activation induces ROS formation in MEL cells and that this process may be involved in events downstream of P2X7 activation, other than apoptosis, in erythroid cells.