Lipid rafts are cholesterol-enriched microdomains of the cell membrane and play an important role in the regulation of membrane proteins. A number of ion channel subunits have been found in lipid rafts in heterologous expression system as well as in cardiomyocytes. Here we investigate whether K⁺ channel subunits are localised in lipid rafts in rat left ventricular myocytes and describe the impact of membrane cholesterol depletion on cardiac K⁺ currents. Left ventricular myocytes were enzymatically isolated from the left ventricular free wall of Wistar rats. Whole cell protein was analysed by sucrose gradient density centrifugation followed by western blot. The ion channel a-subunit Kv4.2, underlying the transient outward K⁺ current (I_to) as well as its b-subunit KChIP2 were partially detected in low density fractions corresponding to lipid rafts. Similarly, a fraction of the delayed rectifier a-subunit Kv1.5 was localised in the low density fractions. Kv2.1, however, was found in the high density fractions only. Corresponding K⁺ currents were assessed by the ruptured-patch whole-cell patch-clamp technique. 20mM methyl-beta-cyclodextrin (MbCD) was used to remove cholesterol from the cell membrane and to destroy lipid rafts. Cholesterol-saturated MbCD (20mM) served as control. MbCD treatment resulted in a 30% decrease in I_to density (from 28.52.0pApF^-1 to 20.01.6pApF^-1, n=15, p<0.001, V_Pip=40mV) within 5min. Treatment with control solution resulted in a smaller, but significant reduction in I_to (from 22.42.3pApF^-1 to 19.12.3pApF^-1, n=15, p<0.001, V_Pip=40mV). Upon treatment with MbCD a 33% increase in the non-inactivating current component (I_SS) was detected: at V_Pip=40mV I_SS increased from 7.60.4pApF^-1 to 10.10.6pApF^-1, n=15, p<0.001. This effect was absent in the control solution (from 7.50.7pApF^-1 to 8.30.9pApF^-1, n=15, n.s.). The increase in I_SS was not sensitive to 100mM 4-aminopyridine or 20mM tetraethylammonium, making a contribution of Kv1.5 or Kv2.1 to this current increase unlikely. We conclude that in rat ventricular cardiomyocytes, a fraction of Kv4.2, KChIP2 and Kv1.5, but not Kv2.1 protein is localised in lipid rafts. Membrane cholesterol depletion results in a reduction in I_to and in an increase in a yet unidentified delayed rectifier current component.