Classically the effect of vagus nerve stimulation is considered to be entirely mediated via muscarinic receptor activation. We have previously shown that direct vagus nerve stimulation (VNS) reduces the slope of action potential duration restitution whilst simultaneously protecting the heart against ventricular fibrillation (VF) initiation in the absence of background sympathetic activity (Ng et al, 2007). We have extended this finding to show that this effect is mediated via nitric oxide (NO) (Brack et al, 2007). In this study our aim was to determine if the protective effects of VNS are dependent on muscarinic receptor activation. Adult male New Zealand rabbits (2.9±0.1Kg, n=9) were used. The isolated heart preparation with intact autonomic nerves was obtained under propofol anesthesia (1 mg/kg, i.v.) as previously described (Ng et al, 2001). The cervical vagus nerves were stimulated at 9.6±1.6Hz, 7.2±1.1V. Ventricular effective refractory period (ERP) was measured with the single extrastimulus method following a 20-beat drive train (300ms cycle length) whilst ventricular fibrillation threshold (VFT) was determined as the minimum current required to induce sustained VF with rapid pacing (30 stimuli x 30ms). ERP and VFT were studied at baseline and during VNS, before and during perfusion with 0.1µM atropine. Data are mean±SEM, whilst statistical analysis was performed using 2-factor repeated measures ANOVA with Bonferroni post tests, P<0.05 was considered significant. During control, VNS decreased heart rate from 147.8±6.5 to 88.9±6.4 bpm (P<0.001) whilst significantly increasing both ERP and VFT from 130.0±6.7 to 147.8±6.7ms (P<0.001) and from 2.2±0.3 to 5.3±0.5mA (P<0.001) respectively. During perfusion with atropine, the effects of VNS on heart rate (143.1±9.8 to 142.2±9.3 bpm, P>0.05) and ERP (137.8±5.7 to 133.3±4.1 ms, P>0.05) were lost whilst the increase in VFT was preserved where VNS increased VFT from 2.0±0.3 to 4.7±0.7mA (P<0.001). These data suggest an acetylcholine / muscarinic receptor independent mechanism underlying the vagal protection of the heart against VF. The context of these findings needs further exploration in relation to the possibility of alternative neurotransmitters and the vagus-NO pathway.