Four hyperpolarization-activated, cyclic nucleotide gated (HCN) channels underlie the pacemaker current I_f in heart and brain. Heart-specific knockout of HCN4, which is highly expressed in the adult sinoatrial node (SAN), lead to embryonal lethality in mice. To determine the role of HCN4 in the adult mouse, we deleted the HCN4 gene in a temporally controlled manner using a tamoxifen-inducible Cre-line that showed efficient expression of Cre-recombinase and corresponding deletion of HCN4 in the SAN. After injection of tamoxifen, double transgenic (HCN4L1/L2; Cre) mice developed a cardiac phenotype within 4 weeks. I_f in isolated SAN cells was reduced by ~80% compared to controls and the activation kinetics of this residual I_f resembled HCN2 which is also expressed in wild type hearts at a low level. Telemetric ECG recordings from HCN4 knockout mice revealed a severe sinus node dysfunction with frequent, sometimes periodically occurring asystolic phases. The pauses in cardiac electrical activity were characterized by the omittance of complete ECG-complexes which where otherwise normal, suggesting improper excitation of the SAN but no primary AV conduction block. Pharmacological inhibition of the residual I_f resulted in aggravation of the condition with prolonged sinus arrests.