To investigate the role of the hyperpolarization-activated current (If) on the subsidiary pacemaker activity of the heart, we studied the effects of an If inhibitor, zatebradine, and a L-type Ca channel inhibitor, verapamil, on the atrioventicular (AV) junctional rhythm and other cardiac responses to stimulation of the left ansa subclavia (LS) in the autonomically decentralized heart of open-chest anesthetized dog. AV junctional rhythm was induced by clamping the sinoatrial (SA) pacemaker region and confirmed by His-bundle electrograms. Zatebradine 0.1 and 0.3 mumol/kg intravenously (i.v.) similarly attenuated both the AV junctional rate and the increase in the junctional rate in response to LS. However, higher doses (1 and 3 mumol/kg i.v.) of zatebradine changed the junctional rhythm to the atrial rhythm in all cases and decreased the atrial rate further but not the increases in the atrial rate in response to LS. On the other hand, verapamil (0.06, 0.2, and 0.6 mumol/kg i.v.) attenuated the increase in the junctional rate induced by LS dose dependently but did not affect the junctional rate itself. After verapamil, zatebradine did not shift the pacemaker site during LS and it further attenuated the positive chronotropic response. These results demonstrate that zatebradine, an If inhibitor, inhibits subsidiary pacemaker activities as well as SA nodal pacemaker activity in situ and suggest that an inhibition of If alters the pacemaker sites due to the different participation of If among the cardiac pacemaker cells.