Nitric oxide (NO) is synthesized from L-arginine through the activity of the synthetic enzyme, NO synthase (NOS). Previous studies have demonstrated the roles of the three isoforms of NOS, namely endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) in cardiovascular regulation. However, no investigation has been done to study their individual role in modulating cardiovascular responses during static skeletal muscle contraction. In this study, we determined the effects of microdialyzing a specific nNOS antagonist into the rostral (RVLM) and caudal ventrolateral medulla (CVLM) on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex using rats. We hypothesized that the NO modulation of the exercise pressor reflex was largely influenced by specific nNOS activity within the ventrolateral medulla. Bilateral microdialysis of a selective nNOS antagonist, 1-(2-trifluoromethylphenyl)-imidazole (1.0 microM), for 30 or 60 min into the RVLM potentiated cardiovascular responses and glutamate release during a static muscle contraction. Levels of GABA within the RVLM were decreased. The cardiovascular responses and neurochemical changes to muscle contraction recovered following discontinuation of the drug. In contrast, bilateral application of the nNOS antagonist into CVLM attenuated cardiovascular responses and glutamate release during a static muscle contraction, but augmented GABA release. These results demonstrate that nNOS in the ventrolateral medulla plays an important role in modulating glutamatergic/GABAergic neurotransmission that regulates the exercise pressor reflex, and contributes to the sympathoexcitatory and sympathoinhibitory actions of NO within the RVLM and CVLM, respectively.