Prolonged cardiac sympathetic nerve activation releases slowly diffusing co‐transmitters such as Galanin (Gal). We tested the hypothesis that Gal acts on local vagal neurons to reduce acetylcholine release and vagal bradycardia.The GalR1 receptor was immunofluorescently co‐localized with choline acetyl‐transferase containing neurons in guinea pig right atria. The effect of Gal was established on the heart rate (HR) response to vagus nerve stimulation (VNS) in isolated guinea pig sinoatrial node/right vagus nerve preparations and also on ³H‐acetylcholine release from right atria during field stimulation. Gal (n=6) reduced the HR response to VNS at 1, 3 and 5Hz (significant at ≥100nM, p<0.05) but not to the stable analogue of acetylcholine, carbamylcholine (n=6). Gal (500nM, n=5) also significantly attenuated the release of ³H‐acetylcholine during field stimulation. The effect of Gal (100nM) on vagal bradycardia was abolished by the GalR1 antagonists galantide (100nM, n=5) and M40 (100nM, n=4) and also by the protein kinase (PK) C inhibitor calphostin C (100nM, n=5). The PKA inhibitor H89 (500nM, n=5) did not prevent the effect of Gal on vagal bradycardia. These results show that GalR1 is located on atrial cholinergic neurons and that Gal attenuates vagal bradycardia via a reduction in acetylcholine release mediated by a GalR1, PKC dependent pathway.Supported by the British Heart Foundation