GABAA receptors generate both phasic and tonic forms of inhibition. In hippocampal pyramidal neurons, GABAA receptors that contain the 5 subunit generate a tonic inhibitory conductance. The physiological role of this tonic inhibition is uncertain, although 5GABAA receptors are known to influence hippocampal-dependent learning and memory processes. Here, we provide evidence that 5GABAA receptors regulate the strength of the depolarizing stimulus that is required to generate an action potential in pyramidal neurons. Neurons from 5 knockout (5-/-) and wildtype (WT) mice were studied in brain slices and cell cultures prepared using whole-cell and perforated-patch clamp techniques. Membrane resistance was 1.6-fold greater in 5-/- than in WT neurons, but the resting membrane potential and chloride equilibrium potential were similar. Membrane hyperpolarization evoked by an application of exogenous GABA was greater in WT neurons. Inhibiting the function of 5GABAA receptor with non-selective (picrotoxin) or 5 subunit-selective (L-655,708) compounds depolarized WT neurons by approximately 3 mV, whereas no change was detected in 5-/- neurons. The depolarizing current required to generate an action potential was 2-fold greater in WT than in 5-/- neurons whereas the slope of the input-output relationship for action potential firing was similar. We conclude that shunting inhibition mediated by 5GABAA receptors regulates the firing of action potentials and may synchronize network activity that underlies hippocampal-dependent behavior.