Neonatal maternal separation (MS) in the rat increases the vulnerability to stressors later in life. In contrast, brief handling (H) in early life confers resilience to stressors in adulthood. Early life programming of stress reactivity may involve the medial prefrontal cortex (mPFC), a region which modulates various stress responses. Moreover, hemispheric specialization in mPFC may mediate adaptive coping responses to stress. In the present study, neuronal activity was examined simultaneously in left and right mPFC in adult rats previously subjected to MS, H or animal facility rearing (AFR). In vivo electrophysiology, under isoflurane anesthesia, was used to conduct acute recordings of unit and local field potential (LFP) activity in response to systemic administration of N-methyl-beta-carboline-3-carboxamide (FG-7142), a benzodiazepine receptor partial inverse agonist which mimics various stress responses. MS decreased basal unit activity selectively in right mPFC. Basal LFP activity was reduced with MS in left and right mPFC, compared to AFR and H, respectively. Hemispheric synchronization of basal LFP activity was also attenuated by MS at lower frequencies. FG-7142 elicited lateralized effects on mPFC activity with different early rearing conditions. Activity in left mPFC was greater with AFR and MS (AFR>MS), whereas activity was predominantly greater with H in right mPFC. Finally, compared to AFR, MS reduced and H enhanced hemispheric synchronization of LFP activity with FG-7142 treatment in a dose-dependent manner. These results indicate that functionally-relevant alterations in mPFC GABA transmission are programmed by the early rearing environment in a hemisphere-dependent manner. These findings may model the hemispheric specialization of mPFC function thought to mediate adaptive coping responses to stressors. They also suggest the possibility that early environmental programming of hemispheric functional coupling in mPFC is involved in conferring vulnerability or resilience to stressors later in life.