In the present study, we have examined the physiological properties of synaptic currents mediated by GlyRs and GABAARs after culturing spinal neurons with a gephyrin antisense oligonucleotide. Application of gephyrin antisense, but not the sense, reduced the glycinergic mIPSC amplitude ( approximately 50%) and frequency ( approximately 85%), indicating the importance of gephyrin for GlyR anchoring at postsynaptic sites. On the other hand, the glycine-evoked current amplitude was unchanged indicating that functional GlyRs were still located in the extrasynaptic membrane. The analysis of the GABAergic transmission in the same neurons revealed approximately 70% reduction in the frequency of the GABAergic mIPSCs, without changes in the amplitude. Interestingly, the modulation of remaining GABAAR-mediated synaptic events by zinc and diazepam was significantly altered by the antisense. These results indicate that gephyrin is required for the membrane insertion/stabilization of the GABAAR gamma2 subunit as well as for its subsequent localization in the postsynaptic membrane.