The great majority of callosal fibers are glutamatergic and excitatory. Anatomical studies have shown that during development almost 20% (0.7-5% in adult) of the fibers are GABAergic (Riederer et al 2004). However, functional properties of GABAergic neurons involved in the callosal-cortical circuits are unknown. Our aim is to elucidate the role of layer VI neurons, including GABAergic interneurons, in the callosal-cortical network. Rats (14 to 24 days-old) were deeply anaesthetized using isoflurane, humanely killed by decapitated, and coronal acute slices, containing bilateral retrosplenial agranular (RSA) cortex and interhemispheric callosal fibers, were prepared. Whole-cell patch-clamp recordings were performed with pipettes containing K-gluconate and biocytin to reconstruct cellular morphology. Electrical stimulation of the corpus callosum evoked heterogeneous synaptic responses recorded in voltage-clamp. Inward currents were evoked at −65mV with the following features: 20-80% rise time=0.39-2.05ms, decay=0.67-5.94ms, latency=4-9.4ms (n=20), and were blocked by the AMPA/kainate antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX, 20 μM). Two cells responded with outward currents only, evoked at −50mV. The currents blocked by the GABA_A antagonist SR95531 (4 μM), and three cells with inward/outward currents sequence. The EPSCs and IPSCs were likely to be monosynaptic, since they had low jitter (range: 0.11-0.73ms), and did not display failures. With Cs⁺-based pipette solution, a slower NMDA receptor-mediated EPSC was detected in the presence of 0.5μM DNQX and 0.5μM SR95531, with a maximum amplitude at −10mV, that was abolished by 40μM AP5. Furthermore, stimulation of the callosal fibers also evoked back-propagating action potentials when neurons sending axons through the corpus callosum were recorded (n=14). Importantly, most of the recorded cells had intrinsic membrane properties and anatomical features indicative of GABAergic interneurons. Anatomically reconstructed cells had an interneuron-like shape with a small fusiform, bipolar soma, one apical dendrite and few dendritic branches. The cell firing was quite heterogeneous, often typical of cortical GABAergic interneurons (Gupta et al, 2003), consisting of fast-spiking accomodating and non-accomodating neurons, as well as stuttering types. We also recorded from regular spiking layer V-VI pyramidal cells, where stimulation of the callosal fibers evoked EPSCs with slower kinetics (range: 20-80%rise time=1.43-2.83ms, decay=10.17-18.9ms, latency=6-8.8ms, n=3). Our data suggest that putative GABAergic interneurones are directly involved in the callosal-cortical network.