Ephrins are guidance cues that modulate axonal growth and the subsequent axonal topographic maps in many regions of the CNS. Here we studied the functional roles of ephrin-A/EphA interactions in the layer-specific pattern of axonal projections in the hippocampus by disrupting the ephrin-A signaling by over-expression of a soluble EphA receptor. Tracing experiments in EphA5-Fc over-expressing mice revealed that reduction of ephrin-A/EphA interactions did not affect the proper distribution of the main hippocampal afferents, i.e. entorhinal and commissural projections. However, further ultrastructural analyses showed a reduction in the density of synaptic terminals in the entorhinal and commissural termination layers in these mice. In addition, using anti-calbindin antibodies, we analyzed the dentate mossy fiber projections following disruption of ephrin-A/EphA interactions throughout developing hippocampus. While the main mossy fiber bundle appeared normal, the infrapyramidal bundle formed longer projections that established ectopic contacts in these transgenic mice. Later, the expected specific pruning of the infrapyramidal bundle was not observed at adult stages. Ultrastructural analyses confirmed a higher number of mossy fiber terminals in the infrapyramidal bundle in adult EphA5-Fc transgenic mice and showed that these terminals were larger and established a greater number of contacts than in controls. Our results demonstrate that ephrin-A/EphA interactions regulate the synaptogenesis of hippocampal afferents and the proper development and refinement of granule cell projections.