While stressors are known to increase medial prefrontal cortex (PFC) glutamate (GLU) levels, the mechanism(s) subserving this response remain to be elucidated. We used microdialysis and local drug applications to investigate, in male Long-Evans rats, whether the PFC GLU stress response might reflect increased interhemispheric communication by callosal projection neurons. We report here that tail-pinch stress (20 min) elicited comparable increases in GLU in the left and right PFC that were sodium and calcium dependent and insensitive to local glial cystine-GLU exchanger blockade. Unilateral ibotenate-induced PFC lesions abolished the GLU stress response in the opposite hemisphere, as did contralateral mGlu(2/3) receptor activation. Local dopamine (DA) D(1) receptor blockade in the left PFC potently enhanced the right PFC GLU stress response, whereas the same treatment applied to the right PFC had a much weaker effect on the left PFC GLU response. Finally, the PFC GLU stress response was attenuated and potentiated, respectively, following alpha(1)-adrenoreceptor blockade and GABA(B) receptor activation in the opposite hemisphere. These findings indicate that the PFC GLU stress response reflects, at least in part, activation of callosal neurons located in the opposite hemisphere and that stress-induced activation of these neurons is regulated by GLU-, DA-, norepinephrine-, and GABA-sensitive mechanisms. In the case of DA, this control is asymmetrical, with a marked regulatory bias of the left PFC DA input over the right PFC GLU stress response. Together, these findings suggest that callosal neurons and their afferentation play an important role in the hemispheric specialization of PFC-mediated responses to stressors.