Four hyperpolarization-activated, cyclic nucleotide-gated cation channel subunits (HCN1-4) have been identified for the current termed I_h (hyperpolarization-activated current) in the brain and I_f (‘Funny’ pacemaker current) in the heart (Stieber et al. 2004). We examined the expression of HCN1, HCN2 and HCN4 by immunolabelling specific Abs against these isoforms in mouse sinoatrial (SA) node. Hearts were excised from 20-25 g adult C57BL mice after schedule 1 killing by cervical dislocation. After dissection of SA node and some surrounding atrial muscle, the preparation (endocardial surface up) was fixed in a tissue bath and was superfused with Tyrode solution at 37°C at a rate of ~4 ml/min through a heat exchanger into the chamber. Extracellular potentials were recorded as described by Lei et al (2004). After electrical mapping, tissue was frozen and sectioned for immunohistochemical studies as described by Lei et al (2004). 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 SA node, the effect of 0.5 and 2 mM Cs⁺(a specific I_f blocker) on spontaneous pacemaker activity of intact SA node/atrium preparations was first examined. 05 mM Cs⁺ prolonged cycle length (CL) of spontaneous beating of SA node/atrium preparations by 15±4 % from 164±26 ms under the control condition to 190±37 ms in the presence of 0.5mM Cs⁺ (n=6, p<0.01). 2 mM Cs⁺ caused a further prolongation of CL by 39±20% (n=6, p<0.01). After 5 to 10 min washing off Cs⁺, spontaneous sinus rhythm fully recovered. Immunostaining of Abs against HCN isoforms is carried out on adjacent sections taken from the same SA node/atrium preparation. HCN1 shows no labelling in the SA node but presents in the control brains tissue sections. HCN2 shows some sporadic extracellular labelling in the SA node. HCN4 shows the cell membrane labelling only in the nodal cells. An Ab against low molecule neurofiliament (NF68) (Dobrzynski et al., 2003) is also tested on these tissue sections, SA node cells are negative to NF68, while the neuron fibbers and neuron body between the SA nodal cells and a ganglia near SA node are positive to NF68. The labelling pattern and expression of NF68 and HCN2 are similar, which suggests that HCN2 expresses preferentially in the neurons in the SA node region. Based on these results, we suggest that HCN4 is the major isoform to contribute to I_f current in mouse SA node pacemaker cells.