In sino-atrial and atrio-ventricular nodal cells, hyperpolarization-activated cyclic nucleotide-gated (HCN) inward current carrying cationic channels, I(f), are expressed that contribute importantly to the diastolic depolarization critical for cardiac pacemaker activity. Although previous studies have demonstrated myocardial expression of both the HCN2 and HCN4 subunits, the specific roles of these subunits in the generation of functional myocardial I(f) channels remain unclear. To explore the molecular compositions of functional cardiac I(f) channels, antibodies targeted against specific C- and N-terminal sequences in HCN2 and HCN4 were exploited to examine HCN2 and HCN4 subunit expression in adult (mouse) heart and to immunoprecipitate endogenous HCN-encoded cardiac I(f) channel complexes. Western blot experiments revealed that although the full-length HCN2 (105 kDa) and HCN4 (160 kDa) proteins are readily detected in transiently transfected HEK-293 cells and in adult (mouse) brain, the molecular mass of the HCN2 protein in the myocardium is approximately 60 kDa. In addition, the myocardial 60-kDa HCN2 protein lacks the C terminus, which contains the cAMP binding domain. In heterologous cells, the C-terminal-truncated HCN2 protein co-assembles with HCN4 to form functional heteromeric HCN channels, which activate faster than homomeric HCN2 or homomeric HCN4 channels, and display properties similar to endogenous myocardial I(f) channels Taken together, these results suggest that functional myocardial I(f) channels reflect the heteromeric assembly of HCN2 and HCN4 subunits and further that the HCN4 subunit underlies the cAMP-mediated regulation of cardiac I(f) channels.