We have previously reported that both transferred microglia and microglia-conditioned medium (MCM) potentiated the N-methyl-D-aspatate (NMDA) receptor-mediated synaptic responses in cortical neurons. To elucidate the mechanism underlying the potentiation of NMDA receptor-mediated responses by microglia, we examined the effects of MCM on NMDA-induced inward currents in mechanically dissociated hippocampal CA1 neurons under whole-cell patch recordings. MCM potentiated the amplitude of NMDA-induced currents up to 10-fold in a dose-dependent manner, and this effect of MCM remained even after boiling or cutting off molecules with a molecular mass more than 3 kDa. In the presence of glycine with a concentration sufficient to saturate the NMDA receptor glycine site, MCM failed to further potentiate the NMDA-induced currents. The glycine site antagonist 5, 7-dichrolokynurenic acid, significantly inhibited the effects of MCM. The effect of MCM was still observed even after treatment with D-amino acid oxidase, a D-serine degrading enzyme. On the other hand, MCM had no significant effect on the voltage-dependent Mg(2+) blockade of NMDA receptors. Furthermore, MCM enhanced the formation of the long-term potentiation in the Schaffer collateral pathway-CA1 pyramidal cell synapses. Using a high performance liquid chromatography system, we found the levels of both glycine and L-serine in MCM to be significantly higher than those in the control medium. It was also noted that an increased glycine productivity of microglia was observed in the hippocampus in the acute phase of neuronal injury. These observations strongly suggest that glycine is a major causative molecule released from microglia that potentiates the NMDA-induced currents.