The regulation of gamma-aminobutyric acid (GABA)-mediated spontaneous inhibitory synaptic transmission by endogenously released glutamate was studied in cultured rat hippocampal neurons. After 7 days in vitro (DIV), both spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) could be detected. After 15 DIV, most postsynaptic spontaneous currents occurred as sEPSC/sIPSC sequences when recorded at a holding voltage of -30 mV. In the presence of the glutamate alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtype antagonist LY303070, both the frequency and amplitude of sIPSC were strongly and reversibly reduced. The N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5), had no effect on sIPSC while cyclothiazide strongly increased sIPSC frequency. Under blockade of AMPA receptors, the kainate- and GluR5-selective kainate receptor agonists, (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid) (ATPA) and (S)-5-iodowillardiine (5IWill), induced a large enhancement of the frequency of small-amplitude sIPSC which was blocked by the non-NMDA receptor antagonist, 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX). All of these effects were sensitive to tetrodotoxin (TTX). In the presence of LY303070 and TTX, kainate could induce a small inward current while GluR5 agonists had no effect. In the presence of NMDA and AMPA receptor antagonists, the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC) could restore sIPSC. When NBQX was used as an AMPA antagonist, the stimulatory effect of t-PDC was blocked while the group I metabotropic glutamate agonist, 3,5-dihydroxyphenylglycine (DHPG), induced a strong enhancement of sIPSC. Therefore, both AMPA and kainate receptors can regulate inhibitory synaptic transmission in cultured hippocampal neurons, the former by tonic activation, the latter when the glutamate concentration is increased by impairing glutonate uptake.