Calcium is a key signaling ion for induction of synaptic plasticity processes that are believed to influence cognition. Mechanisms regulating activity-induced increases in neuronal calcium and related synaptic modifications are not fully understood. Moreover, involvement of specific synapses in discrete aspects of spatial learning remains to be elucidated. We used herpes simplex amplicons to overexpress calbindin D28k (CaBP) selectively in dentate gyrus (DG) granule cells. We then examined the effects on hippocampal network activity by recording evoked synaptic responses in vivo and in vitro and analyzing hippocampal-dependent behavior. Relative to Lac-Z- and sham-infected controls, CaBP overexpression increased mossy fiber (MF-CA3) excitatory postsynaptic potentials and reduced paired-pulse facilitation (PPF), suggesting an increase in presynaptic strength. Additionally, CaBP overexpression reduced long-term potentiation (LTP), caused a frequency-dependent inhibition of post-tetanic potentiation (PTP), and impaired spatial navigation. Thus, increasing CaBP levels selectively in the DG disrupts MF-CA3 presynaptic function and impairs spatial cognition. The results demonstrate the power of gene delivery in the study of the neural substrates of learning and memory and suggest that mossy fiber synaptic plasticity is critical for long-term spatial memory. © 2004 Wiley-Liss, Inc.