The effects of C-type natriuretic peptide (CNP) on heart rate and ionic currents were demonstrated by recording the ECG from adult mice and performing voltage-clamp experiments on single sinoatrial (SA) node cells isolated from mouse heart. The selective natriuretic peptide type C receptor (NPR-C) agonist cANF (10–7M) significantly decreased heart rate in the presence of isoproterenol (5 × 10–9M), as indicated by an increase in the R-R interval of ECGs obtained from Langendorff-perfused hearts. Voltage-clamp measurements in enzymatically isolated single pacemaker myocytes revealed that CNP (10–8M) and cANF (10–8M) significantly inhibited L-type Ca2+current [ ICa(L)]. These findings suggest that the CNP effect on this current is mediated by NPR-C. Further support for an NPR-C-mediated inhibition of ICa(L)in SA node myocytes was obtained by altering the functional coupling between the G protein Giand NPR-C. In these experiments, a “Gi-activator peptide,” which consists of a 17-amino acid segment of NPR-C containing a specific Giprotein-activator sequence, was dialyzed into SA node myocytes. This peptide decreased ICa(L)significantly, suggesting that NPR-C activation can result in a reduction in ICa(L)when CNP is bound and the Giprotein pathway is activated. This effect of CNP appears to be selective for ICa(L), because the hyperpolarization-activated current was unaffected by CNP or cANF. These results provide the first demonstration that CNP has a negative chronotropic effect on heart rate and suggest that this effect is mediated by selectively activating NPR-C and reducing ICa(L)through coupling to Giprotein.