In vitro tract tracing allowing for continuous observation of the perforant path is a crucial prerequisite for experimental studies on the entorhinal-hippocampal interaction in an organotypic slice culture containing the entorhinal cortex, the perforant path, and the dentate gyrus (OEHSC). We prepared horizontal slices of the temporal entorhinal-hippocampal region of the rat on a vibratome, and the perforant path axons were traced by application of the fluorescent tracer Mini Ruby on the entorhinal cortex. After 2 days in vitro (div), the perforant path became visible in most cultures. Entorhinal neurons and single perforant fibers could be followed to the outer molecular layers of the dentate gyrus by in vitro fluorescence microscopy and it was possible to monitor the perforant path directly over a period of 25 div. Moreover, ultrastructural analysis proved the existence of traced perforant path boutons forming synapses with spines and dendritic shafts in the outer molecular layers of the dentate gyrus. Transsection of the prelabelled perforant path in vitro resulted in anterograde degeneration and subsequent phagocytosis of axonal material by activated microglial cells in the zone of denervation. In conclusion, in vitro tracing demonstrates the maintenance of the entorhinal-hippocampal pathway in OEHSCs and permits monitoring of dynamic changes in the prelabeled perforant path after various lesion paradigms, e.g., transsection or neurotoxin treatment. This approach permits further studies on the efficacy of neuroprotectants, cytokines, and growth factors in the treatment of lesion-induced neuronal degeneration.