The aim of this study was to determine whether postnatal mammalian central neurons retain the capacity for axonal regeneration across a lesion site in organotypic cultures of the auditory midbrain. Brain slices from the gerbil inferior colliculus (IC) were obtained from postnatal 6–8 d old animal and cultured for 6–15 d in vitro. IC explants containing an intact commissural projection exhibited robust axonal and dendritic morphologies as assessed with biocytin labelling. In transected explants, the two lobes of the inferior colliculus were cut at the midline and then reapposed to one another in vitro. There was a robust regeneration of commissural fibers across the lesion site in 78% of the biocytin-labeled explants. Massive axonal regeneration was also revealed by immunostaining explants for Tau (100% of sections), an axon-specific microtubule-associated protein. Ultrastructural analyses demonstrated that biocytin-labeled regenerating fibers established de novo synaptic profiles in the contralateral lobe of the inferior colliculus. Finally, the distribution of astrocytes and oligodendrocytes were assessed by staining for glial fibrillary acidic protein (GFAP) and myelin- associated glycoprotein (MAG), respectively. GFAP-positive astrocytes were more widely distributed than in vivo, and oligodendrocytes remained immature, and evenly distributed in all explants. Taken together, these data demonstrate that the postnatal mammalian auditory midbrain can be maintained in vitro, and that central axons are capable of regenerating across the site of injury without the aid of an artificial substrate.