Microglial proliferation and activation have been reported to occur after several central nervous system injuries. In this study, we tested the effects of adenosine triphosphate (ATP) on cultured microglia obtained from the spinal cord of rat embryos. The amounts of tumor necrosis factor α (TNF-α), interleukin 1β and interleukin 6 released from the microglia, which were stimulated by lipopolysaccharide (LPS; 100 ng/ml), were inhibited by the simultaneous addition of ATP in a dose dependent manner (10–300 μM). We examined the effect of several endogenous purines (ATP, ADP, CTP, UDP, UTP) and P 2y receptor agonists (ADPβS and 2-methylthio-ATP) on LPS-induced TNF-α release. The rank order of inhibitory potency of endogenous purines on TNF-α release was: ATP>ADP>>UTP>UDP>CTP. The latter three were much less potent than the former two. The addition of 10 μM 2-methylthio-ATP showed a potency similar to 100 μM ATP. The addition of ADPβS, however, showed less effect. These endogenous purines and selective ATP receptor agonists showed a similar inhibitory effect in their rank order on LPS-induced interleukin 6 release. We demonstrate that ATP inhibits cytokine release from LPS-activated microglia via metabotropic receptors. The application of P 2y receptor agonists might be considered as a pharmacological treatment of several pathological conditions of the spinal cord, including toxic immunoreactions.