Abstract: Brain microglia are a major source of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), which have been implicated in the progression of neurodegenerative diseases. Recently, microglia were revealed to be highly responsive to ATP, which is released from nerve terminals, activated immune cells, or damaged cells. It is not clear, however, whether released ATP can regulate TNF-α secretion from microglia. Here we demonstrate that ATP potently stimulates TNF-α release, resulting from TNF-α mRNA expression in rat cultured brain microglia. The TNF-α release was maximally elicited by 1 mM ATP and also induced by a P2X7 receptor-selective agonist, 2′- and 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate, suggesting the involvement of P2X7 receptor. ATP-induced TNF-α release was Ca2+-dependent, and a sustained Ca2+ influx correlated with the TNF-α release in ATP-stimulated microglia. ATP-induced TNF-α release was inhibited by PD 098059, an inhibitor of extracellular signal-regulated protein kinase (ERK) kinase 1 (MEK1), which activates ERK, and also by SB 203580, an inhibitor of p38 mitogen-activated protein kinase. ATP rapidly activated both ERK and p38 even in the absence of extracellular Ca2+. These results indicate that extracellular ATP triggers TNF-α release in rat microglia via a P2 receptor, likely to be the P2X7 subtype, by a mechanism that is dependent on both the sustained Ca2+ influx and ERK/p38 cascade, regulated independently of Ca2+ influx.