Aims: The cholesterol content of the plasma membrane plays an important role in the regulation of membrane proteins and their localisation in membrane compartments, such as lipid rafts. Previously, we have shown that cholesterol depletion decreases the transient outward K⁺ current (I_to) and increases the delayed rectifier current. The aim of the present study was to investigate the role of cholesterol in the localisation of Kv channel subunits in rat heart tissue and in isolated left ventricular myocytes. Methods: Heart tissue was dissected and ventricular myocytes were enzymatically isolated from the left ventricular free wall of Wistar rats. For cholesterol depletion, tissue or cells were incubated with 20 mM methyl-beta-cyclodextrin (MBCD), while cholesterol-saturated MBCD (20 mM) or Mannitol (20 mM) served as controls. After solubilisation in Triton X-100 (4°C), proteins were analysed by sucrose gradient density centrifugation followed by western blot analysis with antibodies against Kv channel proteins and membrane compartment markers. Results: Under control conditions a fraction of the ion channel a-subunit Kv4.2, underlying I_to as well as its accessory subunit KChIP2 and a fraction of the delayed rectifier a-subunit Kv1.5 located together with lipid raft markers in low density fractions in both, ventricular tissue and in isolated myocytes. Kv2.1, however, was found together with clathrin in the high density fractions only. Depletion of cholesterol from the cell membrane with 20 mM MBCD shifted the localisation of channel proteins Kv4.2, Kv1.5 and KChIP2 to heavier fractions without affecting of Kv2.1 localisation. Conclusion: We conclude that cholesterol content affects the localisation of Kv4.2, KChIP2 and Kv1.5, but not Kv2.1 protein in rat ventricular tissue and in isolated cardiomyocytes.