Abstract The understanding of the mechanisms of a functional synaptic plasticity in the hippocampus has been expanded greatly by the use of in vitro slice preparations. The question addressed in the present study was whether morphological plasticity observed in vivo can also be reproduced in hippocampal slices. In vivo, hippocampal commissural and association fibers are known to sprout and occupy synaptic sites vacated by deafferentation of the dentate gyrus (DG). In hippocampal slice preparations, the major input to the DG is eliminated, so that the DG is deafferented. Might intrinsic neurons sprout to the DG if the slice preparation is maintained for weeks? In this study hippocampal slices obtained from 6-day-old rats were cultured. Stimulation of the dentate stratum moleculare produced antidromic field potentials in the CA3 of the slices cultivated for more than 1 week. The antidromic response was not observed in CA1 pyramidal neurons. The CA3 to DG projection response was also observed in a CA3 mini-slice placed near a co-cultured whole hippocampal slice, when the DG in the latter was stimulated. Moreover, stimulation of the CA3 mini-slice induced synaptic responses in the DG of the whole-slice. The conclusion drawn is that deafferentation could induce axonal sprouting in a neuron-specific manner in hippocampal organotypic culture. This preparation would be potentially useful for the screening of chemical factors that influence sprouting of central neurons.