GABAergic interneurons in the spinal cord display late embryonic and early postnatal expression and repression in accordance with network operation. We investigated the dynamic distribution of GABAergic neurons together with their excitability at different embryonic stages by using mouse organotypic spinal cultures at 8 and 14 days in vitro (DIV). By immunocytochemistry for the GABA-synthesizing enzyme (GAD67), we showed that at 8 DIV, GAD67-labeled neurons were numerous and evenly distributed along the ventral/dorsal axis of the spinal slice. At 14 DIV, GAD67-positive neurons decreased of more than four-fold and displayed a clear dorsal distribution. Next we addressed the temporal expression of ERG, a potassium conductance expressed only transiently by developing spinal interneurons. At 8 DIV ERG-positive cells were numerous, and scattered throughout the slice. As observed for GAD67-positive cells, at 14 DIV a five-fold decrease in ERG positive neurons was accompanied by a dorsal to ventral gradient. Confocal microscopy analysis of simultaneous immunostaining with anti-GABA and anti-ERG antibodies revealed that ERG-positive cells represented mainly GABAergic interneurons. Patch clamp recordings confirmed these data, showing that ventral interneurons expressed functional ERG currents [I_K(ERG)] only transiently. We next examined the role of the I_K(ERG). In organotypic cultures, only ventral interneurons with fast adaptation and GABA-immunoreactivity, and only after one week in culture, were transformed into high frequency bursters by E4031, a selective inhibitor of I_K(ERG), These data suggest that, during an early stage of spinal cord development, the excitability of GABAergic ventral interneurons depended, at least in part, on the function of I_K(ERG).