1. In this paper we have determined the different signalling pathways involved in muscarinic acetylcholine receptor (AChR)-dependent inhibition of contractility in rat isolated atria. 2. Carbachol stimulation of M2 muscarinic AChRs exerts a negative inotropic response, activation of phosphoinositide turnover, stimulation of nitric oxide synthase and increased production of cyclic GMP. 3. Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase, shifted the dose-response curve of carbachol on contractility to the right. These inhibitors also attenuated the muscarinic receptor-dependent increase in cyclic GMP and activation of nitric oxide synthase. In addition, sodium nitroprusside, isosorbide, or 8-bromo cyclic GMP, induced a negative inotropic effect, increased cyclic GMP and activated nitric oxide synthase. 4. These results suggest that carbachol activation of M2 AChRs, exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositides turnover via phospholipase C activation. This in turn, triggers cascade reactions involving calcium/calmodulin and protein kinase C, leading to activation of nitric oxide synthase and soluble guanylate cyclase.