HCN pacemaker channels play an important role in generating and regulating rhythmicity of special neuronal and cardiac cells. They are activated by hyperpolarizing voltages and modulated by the binding of cyclic nucleotides. Four isoforms, HCN1-HCN4, have been identified. Heterotetrameric HCN2/4 channels in Xenopus laevis oocytes show two changes compared to each of the homotetramers: V_1/2 is shifted to more depolarized voltages and activation kinetics is faster (Zhang et al., Biochim Biophys Acta, 2009). However, little is known about the ligand dependence of these channels. Herein, we studied the differences in activation gating of HCN2/4 channels in comparison to the respective homotetramers, thereby focusing on the effect of cAMP. Using two-electrode voltage clamp, patch-clamp and confocal patch-clamp fluorometry in Xenopus laevis oocytes, we monitored the cAMP dependence of activation and ligand binding at both steady-state and non-steady state conditions. We found that (1) in HCN2/4 the apparent affinity for cAMP was between that of the two homotetramers, whereas the Hill coefficient was lowest, (2) cAMP accelerates the voltage-induced activation in HCN2/4 only slightly (factor ~2), resembling HCN4, whereas in HCN2 it accelerates activation in a voltage-dependent manner by a factor of up to ~12, (3) the activation kinetics following a cAMP jump to channels pre-activated by voltage was fastest in HCN2/4, and (4) that ligand binding in HCN2/4 and HCN2 was similar. Our results confirm that coinjection of the HCN2 and HCN4 isoforms in Xenopus oocytes leads to heterotetrameric channels. They suggest that in native heart and neuronal cells the formation of heterotetramers leads to pacemaker channels with specific characteristics, thereby fine-tuning the process of pacemaking.