Mossy fibers from dentate gyrus granule cells establish synapses on CA3 pyramidal neurons during the first 3 postnatal weeks in the rat. Mossy fiber synapses are primarily restricted to the stratum lucidum. When examined by Timm stain after 10-14 days in vitro, cultured hippocampal slices from postnatal day 4 rat pups show a similar mossy fiber termination pattern in stratum lucidum. Thus, axon guidance cues used by mossy fibers in vivo appear to be preserved in these cultured slices. Three experimental manipulations were performed on hippocampal slice cultures to examine whether the axon guidance cues used by mossy fibers are developmentally regulated. First, mossy fibers were transected on the day of culture or day 7 in vitro. Mossy fibers transected on either day were able to reestablish their synaptic pattern in stratum lucidum of CA3. Second, dentates and hippocampi of same age or different age were co-cultured. Same age co-cultures (P4 dentates to P4 hippocampi or P11 dentates to P11 hippocampi) showed good mossy fiber reinnervation of stratum lucidum, as did different age co-cultures from P4 dentates to P11 hippocampi. However, P11 dentates to P4 hippocampi co-cultures showed little mossy fiber reinnervation of stratum lucidum. Third, new P4 or P11 dentates were co-cultured onto hippocampal slices in which mossy fibers had been allowed to degenerate. New mossy fibers reinnervated these hippocampi, but did not reestablish their normal synaptic pattern in stratum lucidum. These three experimental manipulations suggest that mossy fiber axon guidance mechanisms are developmentally regulated, and that existing mossy fibers play a role in directing mossy fiber reinnervation of stratum lucidum.