Previous studies have shown that the septohippocampal projection develops in vitro. It remained unclear, however, whether this in vitro projection resulted from axonal regeneration of septal neurons transected by the culture preparation, or from a priori outgrowth of undamaged cells that had not yet sent an axonal process to the hippocampus at the time of tissue preparation. Here we have prepared slice cultures of identified septohippocampal projection neurons that were retrogradely labeled by hippocampal tracer injection prior to slice preparation. We demonstrate that these identified projection neurons survive axotomy caused by culture preparation, are able to regrow an axonal process in vitro, and innervate a co-cultured hippocampal slice.