1. Modulation of NMDA receptors by metabotropic glutamate receptors (mGluRs) in cultured mouse cortical neurons was investigated using whole-cell and single-channel recordings. 2. NMDA whole-cell current was reversibly attenuated by selective mGluR1/5 agonists (S)-3-hydroxyphenylglycine (3HPG; 10-200 microM), (S)-3,5-dihydroxyphenylglyeine (S-DHPG; 100 microM) and other mGluR agonists: (1S,3R)-1-aminocyclopentane-1,3-decarboxylic acid (1S,3R-ACPD; 200 microM), quisqualate (10 microM) and (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I; 100 microM). 3. The attenuation of NMDA current by 3HPG was totally eliminated by the mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG; 500 microM) and by the selective mGluR1/5 antagonist (S)-4-carboxyphenylglycine (4CPG; 300 microM). 4. mGluR2/3 agonists (2S,1'R,2'R'3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; 3 microM), (S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG; 100-200 microM) and (S)-4-carboxyphenylglycine (4CPG; 300 microM) did not reduce NMDA current. 5. The NMDA-induced increase in intracellular free Ca2+ measured by fura-2 Ca2+ imaging was attenuated by 3HPG (300 microM). 6. The suppression of NMDA current by 3HPG was not affected by treatments that altered intracellular Ca2+ or cAMP levels, or by the protein kinase inhibitor, staurosporine (0.1-0.5 microM). 7. The open probability (NPo) of the NMDA receptor channel in excised outside-out patches was attenuated by 3HPG but not by 4C3HPG. This 3HPG effect was blocked by MCPG. 8. The 3HPG-induced reduction of NMDA whole-cell and single-channel currents was prevented by GDP beta S (200-400 microM). Intracellular dialysis of GTP gamma S (100 microM) also reduced NMDA whole-cell current, and rendered irreversible further reduction induced by 3HPG. 9. These data suggest that a selective activation of mGluR1/5 downmodulates the NMDA receptor channel in a membrane-delimited manner, mediated by G proteins, but not by diffusible second messengers.