Pacemaker HCN channels are found in a variety of excitable cells in heart and brain to control membrane potential. Both cAMP and cGMP facilitate HCN2 opening to the same extent, but the former is more potent (1,2). We asked if the greater cAMP potency, based on electrophysiological assay of its effects on channel opening, was due to better binding or to a more sensitive post binding event. Here, we purified the HCN2 C-terminus and measured binding affinity of cAMP and cGMP to the tetrameric form using isothermal titration calorimetry. We show that cGMP binds to the HCN2 C-terminus with negative cooperativity, as does cAMP, but with a lower affinity for single the high affinity site and the three low affinity binding sites. The overall lower affinity of cGMP binding to the HCN2 channel explains its lower potency as compared to that of cAMP. Nevertheless, high affinity binding of cGMP, Kd < 1M, occurs in a range of concentrations that could be relevant in vivo.