GABA (γ-aminobutyric acid) can mediate inhibition via pre- and post/extrasynaptic GABA receptors. In this paper we demonstrate potentially post/extrasynaptic GABAB receptor-dependent tonic inhibition in L2/3 pyramidal cells of rat medial prefrontal cortex (mPFC) in vitro. First, we show via voltage-clamp experiments the presence of a tonic GABAB receptor-dependent outward current in these neurons. This GABABergic current could be induced by ambient GABA when present at sufficient concentrations. To increase ambient GABA levels in the usually silent slice preparation, we amplified network activity and hence synaptic GABA release with a modified artificial cerebrospinal fluid. The amplitude of tonic GABAB current was similar at different temperatures. In addition to the tonic GABAB current, we found presynaptic GABAB effects, GABAB-mediated inhibitory postsynaptic currents and tonic GABAA currents. Second, we performed current-clamp experiments to evaluate the functional impact of GABAB receptor-mediated inhibition in the mPFC. Activating or inactivating GABAB receptors led to rightward (reduction of excitability) or leftward (increase of excitability) shifts, respectively, of the input–output function of mPFC L2/3 pyramidal cells without effects on the slope. Finally, we showed in electrophysiological recordings and epifluorescence Ca2+-imaging that GABAB receptor-mediated tonic inhibition is capable of regulating network activity. Blocking GABAB receptors increased the frequency of excitatory postsynaptic currents impinging on a neuron and prolonged network upstates. These results show that ambient GABA via GABAB receptors is powerful enough to modulate neuronal excitability and the activity of neural networks.