Hippocampal mossy fibers (MFs), axons of dentate granule cells, run through a narrow strip, called the stratum lucidum, and make synaptic contacts with CA3 pyramidal cells. This stereotyped pathfinding is assumed to require a tightly controlled guidance system, but the responsible mechanisms have not been proven directly. To clarify the cellular basis for the MF pathfinding, microslices of the dentate gyrus (DG) and Ammon's horn (AH) were topographically arranged in an organotypic explant coculture system. When collagen gels were interposed between DG and AH slices prepared from postnatal day 6 (P6) rats, the MFs passed across this intervening gap and reached CA3 stratum lucidum. Even when the recipient AH was chemically pre-fixed with paraformaldehyde, the axons were still capable of accessing their normal target area only if the DG and AH slices were directly juxtaposed without a collagen bridge. The data imply that diffusible and contact cues are both involved in MF guidance. To determine how these different cues contribute to MF pathfinding during development, a P6 DG slice was apposed simultaneously to two AH slices prepared from P0 and P13 rats. MFs projected normally to both the host slices, whereas they rarely invaded P0 AH when the two hosts were fixed. Early in development, therefore, the MFs are guided mainly by a chemoattractant gradient, and thereafter, they can find their trajectories by a contact factor, probably via fasciculation with pre-established MFs. The present study proposes a dynamic paradigm in CNS axon pathfinding, that is, developmental changes in axon guidance cues.