Early embryonic cardiomyocytes are spontaneously beating. The hyperpolarization-activated non-selective cation current (If) appears to be involved in its modulation as it is highly expressed at this stage. The spontaneous beating of early embryonic heart cells is slowed by acetylcholine (ACh) and our earlier studies identified a key role for nitric oxide (NO) in the regulation of the voltage dependent L-type Ca2+ current (ICa,L ). Aim of the present study was to clarify whether and via which signalling pathway(s) If was regulated upon muscarinic receptor activation in early embryonic (E9.5 to E11.5) murine cardiomyocytes. We found that the ACh analogue carbachol (CCh, 10 mmol) led in the majority of cells (68%, n=50) to a strong depression of If by 16.3±1.4% (n=34, p<0.01, voltage steps from -35 mV to ?110 mV). This cholinergic inhibition was mediated by the NO/cGMP/PDE 2 signalling pathway as it was largely reversed by superfusion with inhibitors of this signalling pathway. Analysis of the muscarinic signalling in embryonic cardiomyocytes harvested from NOS 2 (-/-) and NOS 3 (-/-) mice revealed that the NOS 3 isoform was responsible for the muscarinic receptor-induced NO production. Thus, muscarinic receptor stimulation depresses If by generating NO via the NOS 3 and the cGMP/PDE type 2 signalling pathway. This suggests NO is a key signalling molecule involved in the regulation of chronotropy of early embryonic heart cells.