Objective: In the present study we analysed structural and functional properties of fast-spiking GABAergic interneurons in layer 4 (L4) of rat barrel cortex. Firstly, L4 interneurons were categorized according to their morphology. Subsequently, synaptic connections with L4 spiny neurons were investigated to evaluate the functional role of these interneurons within the neuronal microcircuits of the barrel cortex. Methods: Intrinsic properties and unitary synaptic currents were recorded from L4 interneurons and spiny neurons in barrel cortex slices using whole-cell patch clamp and simultaneous biocytin fillings. 3D reconstructions of L4 interneurons were made and analysed regarding the axon-barrel and axon-column relationship. Results: We identified a new type of L4 interneuron characterised by a strictly barrel-confined axon which we named L4 'barrel inhibitor' interneuron. It was subsequently the focus of our study. We characterised the GABAergic synaptic connection of the L4 'barrel inhibitor' interneurons onto L4 spiny neurons. This connection showed a very high connectivity rate of 63% and a mean unitary IPSP amplitude of 0.93±0.81 mV (at Vm = -55mV) with a coefficient of variation of 0.49±0.25. It displayed short- term depression and the mean IPSP latency was 0.6±0.1 ms. The reciprocal connectivity rate was very high with 67%. EPSPs at this connection had a mean unitary amplitude of 1.8±1.7 mV and a latency of 1.0±0.3 ms. L4 'barrel inhibitor' neurons made 3.7±1.3 contacts onto the L4 spiny neuron; these were not only perisomatic, but occurred also at distances greater than 100 mm from the soma and on higher order dendrites. Reciprocally connected L4 spiny neurons made 2.1±0.4 excitatory contacts onto L4 interneurons. The newly identified L4 'barrel inhibitor' interneuron subtype is connected to L4 spiny neurons with a high convergence and divergence and thus is likely to play a major role in intra-barrel feed-forward and feed-back inhibition. Supported by the HASB