We have recently reported that C-type natriuretic peptide (CNP) has a positive chronotropic effect in dogs. We further investigated the effect of CNP on canine cardiac functions: 1) in situ, by exploring the effects of isoproterenol (10 µg), angiotensin II (ANG II, 5 µg), and CNP (40 µg) injections (n = 8) on computerized epicardial mapping of atrial activation to detect a shift in pacemaker location; 2) by examining the presence of natriuretic peptide receptor (NPR)-A and -B mRNAs in atrial and nodal tissues using semiquantitative reverse-transcription polymerase chain reaction; 3) in vitro, using spontaneously beating right atrial preparations (n = 6), by recording the transmembrane potentials of sinoatrial node (SAN) cells before and after injection of CNP (25 µg); and 4) by observing the effects of CNP (25 µg) on contractile force of paced isolated right atrial preparations (n = 6). The results indicate that 1) the site of earliest extracellular electrical activation in the SAN remains mostly unchanged in response to CNP, whereas it shifts to the superior region of the SAN after isoproterenol and ANG II injections; 2) NPR-A and -B mRNAs are present in atrial and nodal tissues; 3) CNP significantly increases the maximal rate of diastolic depolarization and decreases the action potential duration at 75 and 90% of repolarization; and 4) CNP significantly increases atrial contractile force. These results suggest that CNP modifies cardiac ionic currents to produce positive chronotropic and inotropic effects by stimulation of NPR-B receptors, located in the SAN region, and that CNP plays a role in the modulation of cardiac function.