Nitric oxide synthase (NOS), necessary for the production of nitric oxide from l-arginine, exists in three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). We have previously demonstrated that blockade of nNOS within the rostral (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulated cardiovascular responses to static exercise [Ishide, T., Nauli, S.M., Maher, T.J., Ally, A., 2003. Cardiovascular responses and neurotransmitter changes following blockade of nNOS within the ventrolateral medulla during static muscle contraction. Brain Res. 977, 80-89]. In this study, we have examined the effects of bilaterally microdialyzing a specific eNOS antagonist into the RVLM and CVLM on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex in anesthetized rats. Bilateral microdialysis of a selective eNOS antagonist, l-N(5)-(1-iminoethyl)ornithine (l-NIO; 10.0 microM) into the RVLM potentiated cardiovascular responses and increased extracellular fluid glutamate levels during a static muscle contraction. At the same time, levels of GABA within the RVLM were decreased. The cardiovascular responses and neurochemical changes to muscle contraction recovered after discontinuation of the drug. In contrast, bilateral application of the eNOS antagonist into the CVLM attenuated cardiovascular responses and glutamate concentrations during a static muscle contraction, but augmented levels of GABA. These results demonstrate that eNOS within the ventrolateral medulla plays an important role in modulating glutamate/GABAergic neurotransmission, that in turn regulates the exercise pressor reflex. The present study provides further evidence of simultaneous sympathoexcitatory and sympathoinhibitory effects of nitric oxide within the RVLM and CVLM involved in the neural control of circulation during static exercise.