Mice lacking prion protein (PrP-null) are resistant to transmissible spongiform encephalopathies. However, the normal functions of this highly conserved protein remain controversial. This study examines whether PrP-null mice develop normal neuronal pathways, specifically the mossy fibre pathway, within the hippocampus. Timm stained hippocampal sections from the PrP-null group had more granules than the controls in: the granule cell layer, the inner molecular layer of the dentate gyrus, and the infrapyramidal region of CA3. This resembles the mossy fibre collateral and terminal sprouting seen in certain epilepsies. The abnormal connectivity might be predicted to promote epileptiform activity, but extracellular electrophysiological recordings from the granule cell layer revealed a reduced excitability in the PrP-null group, both with and without blockade of GABA(A) receptor-mediated inhibition. We propose that reorganization of neuronal circuity is a feature of PrP-null mice.