Conantokin G (Con G), derived from the venom of Conus geographus, is the most characterized natural peptide antagonist targeted to NMDA receptors. Although Con G is known to bind to the glutamate binding site on the NR2 subunit of the receptor, it is unclear whether it can allosterically modulate the function of the receptor through the glycine binding site on the NR1 subunit. The present study was designed to evaluate the action of Con G on NMDA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) and its modulation by glycine in cultured cortical neurons (13-19 days in vitro) using the whole-cell patch clamp technique. Con G inhibited NMDA receptor-mediated spontaneous EPSCs in a concentration-dependent manner. Also, the potency of Con G decreased as a function of time in culture. The inhibition of EPSCs observed following application of Con G in the presence of high (10 µM) and nominal (no added) concentrations of glycine, were not different at 13 days in vitro (DIV). Further, similar results were obtained with experiments on Con G-induced inhibition of NMDA-evoked whole cell currents. The present results indicate that glycine concentrations do not have a direct effect on Con G-induced inhibition of NMDA currents. In addition, age-dependency in the action of Con G on cortical neurons in vitro suggests that this model system would be useful in examining the effects of different agonists/antagonists on native synaptic NMDA receptors.