Antidepressant drugs have been used for decades, but the neurobiological substrate of their efficacy is not completely understood. Although these drugs have well-established effects on monoamines, evidence is emerging that they may also affect other neurotransmitter systems. It has been shown that treatment with a wide range of antidepressants changes the binding characteristics of the N-methyl-D-aspartate type of glutamate receptor. This change is delayed and occurs only in the cortex. The mechanism that triggers it is unknown. We hypothesized that N-methyl-D-aspartate receptor alterations may be due to changes in the dynamics of cortical excitatory amino acid release. Such changes are of particular interest in areas such as the prefrontal cortex, a region involved in stress responses and affected in major depression. We investigated the effects of two antidepressants with different modes of action, imipramine and phenelzine, on glutamate and aspartate outflow in rat prefrontal cortex and striatum. We showed that antidepressants significantly decreased stimulated glutamate outflow. The effect had a rapid onset, was sustained during chronic administration and was only seen in the prefrontal cortex. This change may initiate receptor alterations. Furthermore, if antidepressants can dampen states of hyperglutamatergic activity and the subsequent excitotoxicity, their chronic use may have a considerable neuroprotective potential in major depression.