Important targets for cAMP signalling in the heart are hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels that underlie the depolarizing “pacemaker” current, I_f [1, 2]. We studied the role of I_f in mice, in which binding of cAMP to HCN4 channels was abolished by a single amino-acid exchange (R669Q). Homozygous HCN4^R669Q/R669Q mice die during embryonic development between embryonic day 11.5 and 12 (E11.5-12). Prior to E12, homozygous and heterozygous embryos display reduced basal heart rates (table 1) and show no or attenuated responses to catecholaminergic stimulation. Isoproterenol (1 μM) superfusion increased the rate of isolated hearts from HCN4^+/+ and HCN4^+/R669Q embryos (37.4 ± 4.5 %, n = 8, and 19.8 ± 2.8 %, n = 6, respectively), whereas no increase was observed in hearts from HCN4^R669Q/R669Q embryos (2.8 ± 2.5 %, n = 5). The genotypic differences in heart rate closely relate to differences in the respective I_f current. Electrophysiological studies on cardiomyocytes from HCN4^R669Q/R669Q embryos revealed that the activation curve of I_f is shifted negatively. The voltage of half-maximal activation (V_1/2) in the three genotypes was -82.3 ± 3.2 mV (n = 12) for wild-type, -89.1 ± 6.6 mV (n = 12) for heterozygous, and -95.5 ± 4.5 mV (n = 9) for homozygous cardiomyocytes. Moreover, the V_1/2 in homozygous cardiomyocytes cannot be moved towards more positive values by the addition of cAMP (500 μM). While heterozygous embryos display an obvious cardiac phenotype, adult HCN4^+/R669Q mice display normal heart rates at rest and during exercise. However, following exercise, hearts exhibit pauses and sino-atrial node block. We conclude that in the embryo, HCN4 is one of the principal cardiac pacemakers and persistent elevation of the heart rate by cAMP is essential for viability. In adult mice, HCN4 channels take no longer part in heart rate regulation, but prevent sinus pauses during and after stress. Thus, in mice, the mechanism of pacemaking switches during development and HCN4 may serve two different functions that both critically rely on the presence of cAMP. Data are expressed as mean ± SEM.