Severed adult CNS axons can extend over long distances when a permissive 'milieu', such as grafted Schwann cells or ensheathing cells, is provided. Moreover, functional blocking of endogenous inhibitory factors, such as Nogo-A or proteoglycans, enhances the regeneration of axotomized neurons. Here we examine whether guidance cues available during the development of axonal pathways could also potentiate the regeneration of lesioned adult circuits. The Cajal-Retzius cells in the hippocampus are transient pioneer neurons that guide entorhino-hippocampal afferents to their target layers. By using an in vitro model of axotomy of the entorhino-hippocampal pathway we show that Cajal-Retzius cells triggered the regeneration of the axotomized entorhino-hippocampal pathway. Furthermore, the regrowth induced by Cajal-Retzius cells was robust and its pattern was indistinguishable from that of the unlesioned entorhino-hippocampal pathway. Thus, regenerating axons regrew in a layer-specific fashion towards the appropriate target layers, making synaptic contacts with target pyramidal neurons. Interestingly, the ability of lesioned entorhinal axons to regrow was maintained for at least 9 days after axotomy. These results show that the growth-promoting cells controlling the development of neural circuits will be a relevant approach to promoting the regeneration of lesioned adult CNS pathways.