Hypertension is associated with reduced cardiac vagal activity which is an independent predictor of mortality [1-3]. Neuronal production of nitric oxide (NO) facilitates cardiac parasympathetic transmission although this pathway may be impaired by oxidative stress caused by hypertension [4]. We tested the hypothesis that hypertension attenuates peripheral vagal responsiveness in the spontaneously hypertensive rat (SHR) due to impaired NO-cGMP signalling and gene transfer of neuronal NO synthase (nNOS) can restore vagal function. Cardiac vagal responses in isolated SHR atrial/right vagus preparation are significantly attenuated compared to age-matched normotensive Wistar-Kyoto (WKY) rats (p<0.05, WKY n=7, SHR n=9) at 7Hz, 5Hz and 3Hz. [3H]acetylcholine (ACh) release was also 33.08% lower in SHR compared to the WKY (P<0.01). The NO donor, sodium nitroprusside (SNP), augmented [3H]ACh release in WKY (+0.20±0.09%, p<0.05), whereas the soluble guanylate cyclase (sGC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxaline-1-one (ODQ) attenuated [3H]ACh release in WKY (-0.47±0.11%, p<0.05). No effects were seen in the SHR during nerve stimulation into SNP and ODQ. In contrast, SHR (n=6) were 21.52% higher effect to carbachol with elevated production of cGMP than WKY (n=5, p<0.05). Following gene transfer of nNOS (Ad.nNOS) into the right atria, there was a significantly increased vagal responsiveness in vivo in the SHR compared to transfection with Ad.eGFP. Measurement NOS activity and protein expression of atria indicated an increase of nNOS activity (from 15.24±0.53 to 18.78±1.29 fmol/mg/min, p<0.05), α1-sGC (from 0.28±0.05 to 0.45±0.03, p<0.05) and nNOS protein expression (from 0.81±0.11 to 1.08±0.04, p<0.05) in Ad.nNOS (n=6) -treated WKY atria compared with Ad.eGFP (n=6). These results suggest that a significant component of cardiac vagal dysfunction in hypertension is attributed to an impairment of the pre-synaptic NO-cGMP pathway and that overexpression of nNOS can reverse the neural phenotype.