A sedentary lifestyle is a major risk factor for cardiovascular disease, and both conditions are associated with overactivity of the sympathetic nervous system. Ongoing discharge of sympathetic nerves is regulated by the rostral ventrolateral medulla (RVLM), which in turn is modulated by the primary excitatory and inhibitory neurotransmitters glutamate and -amino-butyric acid (GABA), respectively. We reported previously that sedentary conditions enhance GABAergic modulation of sympathoexcitation in the RVLM, despite overall increased sympathoexcitation. Thus, the purpose of this study was to test the hypothesis that sedentary conditions increase responsiveness to GABA in RVLM. Male Sprague-Dawley rats performed either chronic wheeling running or remained sedentary for 12-15 weeks. Animals were instrumented to perform RVLM microinjections under Inactin anesthesia while recording mean arterial pressure (MAP) and splanchnic sympathetic nerve activity (SSNA). Unilateral microinjections of GABA (30 nl, 0.3-600 mM) into the RVLM produced dose-dependent decreases in MAP and SSNA; however, no group differences were observed. Inhibition of the contralateral RVLM (muscimol, 2 mM, 90 nl) caused decreases in MAP and SSNA that were not different between groups but enhanced decreases in SSNA to GABA in sedentary rats only. In sinoaortic denervated rats, GABA microinjections prior to or after inhibition of the contralateral RVLM caused decreases in MAP and SSNA that were not different between groups. Results suggest that contralateral RVLM plays an important role in buffering responses to inhibition of the ipsilateral RVLM under sedentary conditions. Enhanced sympathoinhibition may act to reduce already elevated sympathetic nervous system activity following sedentary conditions.