P2X(7) receptor subunits form homomeric ATP-gated, calcium-permeable cation channels. In this study, we used Western blots and immunocytochemistry to demonstrate that P2X(7) receptors are abundant on presynaptic terminals of mossy fiber synapses in the rat hippocampus. P2X(7)-immunoreactive protein was detected using a specific P2X(7) antibody in Western blots of protein isolated from whole hippocampus and from a subcellular fraction containing mossy fiber synaptosomes. P2X(7) immunoreactivity was colocalized with syntaxin 1A/B-immunoreactivity in mossy fiber terminals in the dentate hilus and stratum lucidum of CA3. Extracellular and whole-cell voltage-clamp recordings in CA3 revealed that bath application of the potent P2X(7) agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP) caused a long-lasting inhibition of neurotransmission at mossy fiber-CA3 synapses. Consistent with a presynaptic action at mossy fiber synapses, Bz-ATP had no significant effect on neurotransmission at associational-commissural synapses in CA3 but increased paired-pulse facilitation during depression of mossy fiber evoked currents. In addition, Bz-ATP had no postsynaptic effect on holding current or conductance of CA3 neurons. Bz-ATP-induced mossy fiber synaptic depression was blocked by the P2X(7) antagonist oxidized ATP but not by the P2X(1-3,5,6) antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid or the P2Y antagonist reactive blue 2. Finally, an antagonist of p38 MAP kinase activation [4-(4-fluorophenyl)2-(4-methylsulfinylphenyl)5-(4-pyridyl)imidazole] but not extracellular signal-regulated kinase 1/2 MAP kinase (2'-amino-3'-methoxyflavone) blocked the synaptic depression mediated by Bz-ATP, suggesting that this presynaptic inhibition was mediated by activation of p38 MAP kinase. The results of the present study demonstrate that activation of presynaptic P2X(7) receptors depresses mossy fiber-CA3 synaptic transmission through activation of p38 MAP kinase.