We have previously reported that, depending on the dose, nitric oxide (NO)-generating agents exert a dual facilitatory and inhibitory action on glutamatergic transmission on the rostral ventrolateral medulla (RVLM) neurons. The molecular mechanisms underlying the NO-mediated synaptic inhibition have not yet been defined. Here we show that the amplitude of excitatory postsynaptic currents (EPSCs) was reversibly reduced by the NO donors 3-morpholinylsydnoneimine (SIN-1) (1 mM) and spermine NONOate (1 mM). This effect was antagonized by an active peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride, G i/ocoupled receptor blockers, N-ethylmaleimide and pertussis toxin, A 1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, or adenosine deaminase. However, NO-sensitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, GABA B receptor antagonist (2S)-(ϩ)-5,5-dimethyl-2-morpholineacetic acid (SCH50911), or can--pyrazole-3-carboxamide hydrochloride (SR141716A) had no effect on the inhibitory action of SIN-1 on EPSCs. Perfusion of adenosine mimicked and subsequently occluded the action of SIN-1. Inhibition of EPSC amplitude by SIN-1 was associated with an increase in the paired-pulse ratio of EPSCs. Furthermore, SIN reduced the frequency of spontaneous EPSCs without altering their amplitude of distribution. Pretreatment with N-type Ca 2ϩ -channel blocker -conotoxin GVIA selectively blocked SIN-1-induced inhibition of EPSCs. These results suggest that a higher dose of SIN-1 acts presynaptically to elicit a synaptic depression on the RVLM neurons through an inhibition of presynaptic N-type Ca 2ϩ -channel activity, leading to reduced glutamate release. The presynaptic action of SIN-1 is mediated by the formation of peroxynitrite, which subsequently acts to release adenosine to activate A 1 adenosine receptors.Nitric oxide (NO) is a highly diffusible and short-lived gaseous molecule known to be involved in many physiological functions, including central circulatory regulation (Krukoff, 1999;Zanzinger, 1999). One of the potential areas in the central nervous system for NO to exert its modulatory action on cardiovascular function is the RVLM, which contains sympathetic premotor neurons responsible for maintaining the tonic excitation of sympathetic preganglionic neurons involved in cardiovascular regulation (Spyer, 1994;Zanzinger et al., 1995). Consistent with the notion of a pivotal role of NO in regulating sympathetic outflow of the RVLM, several studies using immunohistochemistry, NADPH-diaphorase staining, or autoradiography have shown the presence of neuronal NO synthase (nNOS), inducible NOS (iNOS), and