Glutamate activated NMDA receptors mediate excitatory synaptic transmission in the central nervous system and play an important role in many physiological processes including long term potentiation, synaptogenesis and excitoxicity. The modulation of NMDA-mediated responses by 5-hydroxytryptamine (5-HT) has been reported in spinal cord motorneurones (Chesnoy-Marchais & Barthe, 1996; MacLean & Schmidt, 2001) and this interaction has been implicated in the control generation of motor rhythm activity in the mammalian spinal cord. In the present study, the effect of 5-HT on heteromeric recombinant NMDA receptors (NR1a+2A, NR1a+2B and NR1a+2C) expressed in Xenopus oocytes was investigated using the two-electrode voltage clamp recording technique. Stage V-VI oocytes were removed from anaesthetized female Xenopus laevis in accordance with the guidelines of the University of Queensland Animal Experimentation Ethics Committee. In the absence of external Mg²⁺ ions, 5-HT inhibited NMDA receptor-mediated currents evoked with 100 µM glutamate and 10 µM glycine in a concentration-dependent manner. The inhibitory effect of 5-HT was strongly voltage-dependent whereby 40 mV depolarization shifted the concentration-response curve to the right and increased the half-maximal inhibitory concentration (IC₅₀) approximately ten-fold. The IC₅₀ ′s obtained for 5-HT block of NR1a+2A, NR1a+2B and NR1a+2C subunit combinations expressed in oocytes voltage clamped at −120 mV were 60, 50 and 40 µM, respectively, and increased to 500 µM for NR1a+2A and 300 µM for NR1a+2B and NR1a+2C in oocytes held at −70 mV. Current-voltage curves obtained using voltage ramps applied during steady-state glutamate responses markedly rectified in the presence of 5HT whereby inhibition of glutamate-evoked currents by 5-HT increased with membrane hyperpolarization. The voltage sensitivity of the inhibition (e-fold change per 20 mV change in membrane potential) indicates a block by 5-HT deep within the membrane electric field. The 5-HT precursor, tryptamine inhibited glutamate-evoked currents through NMDA receptors with a similar potency whereas tryptophan (1 mM) failed to inhibit the response. Taken together, these data suggest that 5-HT and related compounds can attenuate glutamate-mediated excitatory synaptic responses and may provide a basis for drug treatment of excitotoxic neurodegeneration.