Gephyrin is a tubulin-binding protein that acts as a scaffold for clustering glycine and GABA(A) receptors at postsynaptic sites. In this study, the role of gephyrin on GABA(A) receptor function was assessed at the post-translational level, using gephyrin-specific single chain antibody fragments (scFv-gephyrin). When expressed in cultured rat hippocampal neurons as a fusion protein containing a nuclear localization signal, scFv-gephyrin were able to remove endogenous gephyrin from GABA(A) receptor clusters. Immunocytochemical experiments revealed a significant reduction in the number of synaptic gamma2-subunit containing GABA(A) receptors and a significant decrease in the density of the GABAergic presynaptic marker vesicular GABA transporter (VGAT). These effects were associated with a slow down of the onset kinetics, a reduction in the amplitude and in the frequency of miniature inhibitory postsynaptic currents (mIPSCs). The quantitative analysis of current responses to ultrafast application of GABA suggested that changes in onset kinetics resulted from modifications in the microscopic gating of GABA(A) receptors and in particular from a reduced entry into the desensitized state. In addition, hampering gephyrin function with scFv-gephyrin induced a significant reduction in GABA(A) receptor-mediated tonic conductance. This effect was probably dependent on the decrease in GABAergic innervation and in GABA release from presynaptic nerve terminals. These results indicate that gephyrin is essential not only for maintaining synaptic GABA(A) receptor clusters in the right position but also for regulating both phasic and tonic inhibition.