Purpose: Implantable cardiac vagus nerve stimulators are a promising new treatment for heart failure, which may improve both quality of life and ejection fraction[1]. Animal studies also suggest an anti-fibrillatory effect of stimulating the cardiac vagus nerve that may involve a nitric oxide (NO) dependent pathway[2], although the exact site of action in the cardiac-neural axis is still debated. We investigated whether carbamylcholine (CCh), a stable analogue of the neurotransmitter acetylcholine, can mimic the effect of vagus nerve stimulation on ventricular fibrillation threshold (VFT), and whether this mechanism is dependent on muscarinic receptor stimulation and/or generation of NO. Methods: Experiments conformed to “Principles of laboratory animal care” (NIH Publication No. 85-23, revised 1996) and the Animals (Scientific Procedures) Act 1986 (UK). Hearts were isolated from adult male Sprague Dawley rats (300-350g) and Langendorff perfused with tyrode solution in constant flow mode (11ml/min, baseline perfusion pressure 54.75±7.27mmHg, LV developed pressure 77.5±5.6mmHg, heart rate 282.8±3.4bpm, n=6). VFT was reproducibly determined by pacing at a fixed cycle length (150msec) for 20 beats followed by a 5sec 50Hz burst at increasing current amplitude (mA) until sustained VF was induced. VF was cardioverted to sinus rhythm by perfusion with 1ml of high concentration potassium chloride solution (50mmol/L). VF could be induced on three successive occasions without a significant change in VFT (n=6). All data is presented as mean±SEM. Results: CCh (200nM, n=9) significantly (p<0.05) reduced baseline heart rate from 292±8 to 224±6bpm). Independent of this heart rate change, CCh also caused a significant increase in VFT in paced hearts similar to vagus nerve stimulation that could be reversed with washout of the drug (control 1.5±0.25 vs. CCh 2.4±0.4 mA vs. wash out 1.14±0.18 mA). The effect of CCh on VFT was completely abolished by atropine (10μM, n=4) or the non-specific competitive NOS inhibitor N-omega-nitro-L-arginine (L-NA 100μM, n=6). The inhibitory action of L-NA could be reversed by the addition of the NOS substrate L-arginine (5mM). The NO donor sodium nitroprusside (SNP 10μM, n=8) mimicked the effects of CCh and significantly increased VFT (baseline 1.4±0.125 vs, SNP 2.9±0.83 vs. wash out 1.18±0.23 mA). Conclusions: These data demonstrate that the protective effect of cardiac vagal nerve stimulation on VFT is mimicked by CCh in the rat. Muscarinic receptor stimulation and the generation of NO appear to be involved in mediating this protective effect, presumably via a pre-synpatic and post synaptic coupled pathway.