gamma-Aminobutyric acid type A (GABA(A)) receptor alpha5 subunits, which are heavily expressed in the hippocampus, are potential drug targets for improving cognitive function. They are found at synaptic and extrasynaptic sites and have been shown to mediate tonic inhibition in pyramidal neurons. We tested the hypothesis that alpha5 subunits also contribute to synaptic inhibition by measuring the effect of diazepam (DZ) on spontaneous and stimulus-evoked inhibitory postsynaptic currents (IPSCs) in genetically modified mice carrying a point mutation in the alpha5 subunit (alpha5-H105R) that renders those receptors insensitive to benzodiazepines. In wild type mice, DZ (1 microM) increased the amplitude of spontaneous IPSCs (sIPSCs) and stimulus-evoked GABA(A,slow) IPSCs (eIPSCs) and prolonged the decay of GABA(A,fast) sIPSCs. In alpha5-mutant mice, DZ increased the amplitude of a small-amplitude subset of sIPSCs (<50 pA) and eIPSCs (<300 pA) GABA(A,slow) and prolonged the decay of GABA(A,fast) sIPSCs, but failed to increase the amplitude of larger sIPSCs and eIPSCs GABA(A,slow). These results indicate that alpha5 subunits contribute to a large-amplitude subset of GABA(A,slow) synapses and implicate these synapses in modulation of cognitive function by drugs that target alpha5 subunits.