The atrioventricular node (AVN) serves not only as the exclusive conduction pathway between the atrium and ventricles, but also as a secondary pacemaker in the event of sinoatrial node (SAN) failure. To investigate the mechanism underlying pacemaking in the mouse AVN, the expression of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels (responsible for I_f current) were studied in the AVN (as well as the SAN). Hearts were excised from humanely killed 20-25 g adult C57BL mice. The AVN with some surrounding tissue was dissected from the right atrioventricular junction at the triangle of Koch. The preparation was fixed in a tissue bath and superfused with Tyrode solution and extracellular potentials were recorded as described by Lei et al. (2004) (1). After electrical recording, tissue was frozen and sectioned, and histological and immunohistochemical studies were carried out as described by Lei et al. (2004) (1). Images were acquired by a Zeiss LSM5 PASCAL confocal microscope and analysed by Image J software. Data are presented as means ± SEM (number of preparations). Differences were evaluated by Student’s t test and a difference was considered significant if p<0.05. To confirm the functional role of I_f in mouse AVN, the effect of 0.5 mM Cs⁺ (blocker of I_f) on the spontaneous pacemaker activity of the isolated AVN preparations was examined. Cs⁺ prolonged the cycle length (CL; time between spontaneous action potentials) by 122% from 252 ± 53 ms under the control conditions to 560 ± 154 ms in the presence of Cs⁺ (n=5, p<0.01). In contrast, Cs⁺ prolonged the CL of the SAN by just 15%. After washing off Cs⁺ for 5 to 10 min, spontaneous AVN rhythm recovered fully. Immunostaining was carried out using antibodies against HCN isoforms. There was no labelling of HCN1 in the AVN and SAN. There was non-myocyte labelling of HCN2 in the AVN and SAN. There was punctate membrane labelling of HCN4 in the myocytes of the AVN and SAN, but the labelling intensity in the AVN was ~20% of that in the SAN. Based on the effect of Cs⁺ on the spontaneous activity of the AVN and SAN (and also preliminary data obtained with the I_f blocker, ZD7288), we conclude that I_f plays a more important role in the pacemaking of the AVN than the SAN. Based on the immunostaining, we conclude that HCN4 is the major isoform responsible for I_f in the mouse AVN and SAN and, paradoxically, HCN4 is more abundant in the SAN than AVN.