The hippocampal formation (HPC) mediates processes associated with learning, memory, anxiety and fear. The glutamate N-methyl-d-aspartate (NMDA)-receptor subtype is involved in many HPC functional processes related to learning and memory. Although not tested for the HPC, NMDA-receptor antagonists reduced fear and anxiety related responses when applied to other brain regions mediating defensive behaviour. Consequently, this study evaluated the effects of ventral or dorsal HPC application of the NMDA-receptor antagonist, AP5, in rats submitted to the Trial 1/Trial 2 elevated plus-maze (EPM) task. Ventral, but not dorsal, infusions of AP5 (6 and 24 nmol) before EPM Trial 1 increased open arms exploration and reduced risk assessment behavior, suggesting an anxiolytic-like effect. Furthermore, no interference in the avoidance responses was detected during EPM Trial 2 after AP5 infusion into the ventral or dorsal HPC before Trial 1, post-trial 1, or before Trial 2. These data support the notion of differential involvement of ventral HPC, but not dorsal, in mechanisms associated with anxiety and suggest the participation of the glutamatergic transmission, through NMDA receptor, into the ventral HPC in the mediation of defensive behavior.