Funny current (I_f), formed by hyperpolarization-activated HCN channels, is supposed to be crucial for membrane clock regulating the cardiac pacemaker mechanism. We examined the presence and activity of HCN channels in the brown trout (Salmo trutta fario) sinoatrial (SA) pacemaker cells, and their putative role in heart rate (fH) regulation. Six HCN transcripts (HCN1, HCN2a, HCN2ba, HCN2bb, HCN3 and HCN4) were expressed in the brown trout heart. The total HCN transcript abundance was 4.0 and 4.9 times higher in SA pacemaker tissue than in atrium and ventricle, respectively. In the SA pacemaker HCN3 and HCN4 were the main isoforms representing 35.8±2.7% and 25.0±1.5%, respectively, of the total HCN transcripts. Only a small I_f with a mean current density of -1.2±0.37 pA/pF at -140 mV was found in 4 pacemaker cells out of 16 spontaneously beating cells examined, despite the optimization of recording conditions for I_f activity. I_f was not found in any of the 24 atrial myocytes and 21 ventricular myocytes examined. HCN4 co-expressed with MiRP1 β-subunit in CHO cells, generated large I_f currents. In contrast, HCN3 (+MiRP1) failed to produce I_f in the same expression system. Cs⁺ (2 mM), which blocked 84±12% of the native I_f, reversibly reduced fH 19.2±3.6% of the excised multicellular pacemaker tissue from 53±5 to 44±5 beats per minute (P<0.05). However, this effect was probably due to the reduction of I_Kr, which was also inhibited (63.5±4.6%) by Cs⁺. These results strongly suggest that fH regulation in the brown trout heart is largely independent on I_f.