The difference in the amounts of NR2 subunits contained in NMDA receptors of the hippocampus has been related to their different involvement in activity-dependent synaptic plasticity. Here, we show that Ro 25-6981, a high-affinity and selective blocker of NMDA receptors containing NR2B subunits, is able to block the acquisition of a trace conditioning paradigm in adult rats, a task that requires the active participation of hippocampal circuits. Reconditioning with the same trace paradigm was also prevented by Ro 25-6981. In addition, we show that the slope of monosynaptic field excitatory postsynaptic potentials evoked at the dentate gyrus by single pulses presented to the medial perforant pathway increases significantly across conditioning sessions and during reconditioning, in a linear relationship with the increase in the number of classically conditioned eyelid responses. Administration of Ro 25-6981 prevented these learning-related changes in synaptic strength at the perforant pathway–dentate granule cell synapse. The present results suggest the involvement of NR2B-containing NMDA receptors in hippocampal functions related to both associative learning and activity-dependent synaptic plasticity.