Objective: We have investigated the properties of the hyperpolarization-activated (I_f) current in pacemaker cells from the mouse sino-atrial node (SAN). Methods: The I_f current was studied in cells isolated enzymatically from the SAN region of adult C57BL6/J mice. The whole-cell variation of the patch-clamp technique was employed to investigate the basic properties of I_f. Results: In mouse SAN cells, the I_f current density at −120 mV was 18±2 pA/pF (n=23). I_f was not detected in cells showing atrial-like morphology that were also found in SAN preparations (n=7). I_f was blocked by 5 mM Cs⁺, was inhibited by application of 5 μM acetylcholine, and was increased by 10 μM noradrenaline. The I_f current reversal potential was −31±2 mV under physiological concentration of Na⁺ and K⁺ ions. Lowering the extracellular Na⁺ concentration reduced I_f amplitude, while increased when the extracellular K⁺ concentration was augmented. I_f voltage for half activation was −87±1 mV (n=6). Conclusions: We conclude that the native I_f current in mouse SAN cells shows functional properties that are similar to I_f described in rabbit SAN tissue. This study opens the possibility of investigating the involvement of I_f in the regulation of heart rate in genetically modified mice.