Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the threshold for action potential generation and integrates excitatory signals. Tonic currents are known to be largely mediated by extrasynaptic γ-aminobutyric acid type A (GABAA) receptors that are persistently activated by submicromo-lar concentrations of ambient GABA. We recently reported that, in cultured hippocampal neurons, the clustering of synaptic GABAAreceptors significantly affects synaptic transmission (Petrini, E. M., Zacchi, P., Barberis, A., Mozrzymas, J. W., and Cherubini, E. (2003)J. Biol. Chem.278, 16271–16279). In this work, we demonstrated that the clustering of extrasynaptic GABAAreceptors modulated tonic inhibition. Depolymerization of the cytoskeleton with nocodazole promoted the disassembly of extrasynaptic clusters of δ and γ2subunit-containing GABAAreceptors. This effect was associated with a reduction in the amplitude of tonic currents and diminished shunting inhibition. Moreover, diffuse GABAAreceptors were less sensitive to the GAT-1 inhibitor NO-711 and to flurazepam. Quantitative analysis of GABA-evoked currents after prolonged exposure to submicromolar concentrations of GABA and model simulations suggest that clustering affects the gating properties of extrasynaptic GABAAreceptors. In particular, a larger occupancy of the singly and doubly bound desensitized states can account for the modulation of tonic inhibition recorded after nocodazole treatment. Moreover, comparison of tonic currents recorded during spontaneous activity and those elicited by exogenously applied low agonist concentrations allows estimation of the concentration of ambient GABA. In conclusion, receptor clustering appears to be an additional regulating factor for tonic inhibition.