Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels generate rhythmicity in specialized neuronal and cardiac cells. They are primarily activated by hyperpolarizing voltages and modulated by binding of the cytosolic ligand cAMP. To learn more about the role of the four subunits in the activation of HCN channels, we simultaneously measured in homotetrameric HCN2 channels, activated by voltage, the time courses of ligand binding and subsequent activation when applying ligand jumps. As ligand we used the functional fluorescent derivative of cAMP, 8-DY547-AET-cAMP (Kusch et al., 2010). Ligand binding was quantified by confocal patch-clamp fluorometry (Biskup et al., 2007), fast solution changes were realized with a piezo-driven double barreled pipette. The time courses of ligand binding and unbinding as well as activation and deactivation at three ligand concentrations were globally fitted with a Markovian state model. It turned out that (1) a sufficient model had to contain four binding steps, that (2) at each degree of liganding a closed-open isomerization is required, and that (3) transitions between neighbored open states are required. As a result we obtained a complete set of rate constants for the liganding of each subunit and all closed-open isomerizations. It is concluded that the ligand-induced enhancement of activation is a complex and highly cooperative process which, depending on the ligand concentration, favors channels with zero, two, or four over those with one or three ligands bound. Though the spatial relation of the subunit interaction remains open, our data provide a detailed view how the subunits interact kinetically.