Lipid rafts are discrete membrane subdomains rich in sphingolipids and cholesterol. In ventricular myocytes a function of caveolae, a type of lipid rafts, is to concentrate in close proximity several proteins of the beta-adrenergic transduction pathway. We have investigated the subcellular localization of HCN4 channels expressed in HEK cells and studied the effects of such localization on the properties of pacemaker channels in HEK and rabbit sinoatrial (SAN) cells. We used a discontinuous sucrose gradient and Western blot analysis to detect HCN4 proteins in HEK and in SAN cells, and found that HCN4 proteins localize to low-density membrane fractions together with flotillin (HEK) or caveolin-3 (SAN), structural proteins of caveolae. Lipid raft disruption by cell incubation with methyl-beta-cyclodextrin (MbetaCD) impaired specific HCN4 localization. It also shifted the midpoint of activation of the HCN4 current in HEK cells and of I(f) in SAN cells to the positive direction by 11.9 and 10.4 mV, respectively. These latter effects were not due to elevation of basal cyclic nucleotide levels because the cholesterol-depletion treatment did not alter the current response to cyclic nucleotides. In accordance with an increased I(f), MbetaCD-treated SAN cells showed large increases of diastolic depolarization slope (87%) and rate (58%). We also found that the kinetics of HCN4- and native f-channel deactivation were slower after lipid raft disorganization. In conclusion, our work indicates that pacemaker channels localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties.