Lipid rafts are cholesterol-enriched microdomains of the cell membrane. Here we investigate the localization of the pore forming K⁺-channel α-subunit Kv4.2 and the β-subunit KChIP2, underlying the transient outward K⁺ current (I _to), in lipid rafts in left ventricular myocytes. Furthermore, we explored the impact of membrane cholesterol depletion (using 20 mM methyl-beta-cyclodextrin (MBCD)) on K⁺ outward currents. Cholesterol-saturated MBCD (20 mM) served as control. Myocytes were isolated from the left ventricular free wall of Wistar rats. The Triton X-100 (4 °C) insoluble fraction of whole cell protein was analyzed by sucrose density gradient centrifugation followed by Western blot. Kv4.2 and KChIP2 were partially detected in low-density fractions (lipid rafts). MBCD treatment (5 min) resulted in a shift of Kv4.2 and KChIP2 towards high-density fractions. K⁺ currents were assessed by whole-cell patch-clamp. MBCD treatment resulted in a 29 ± 3 % decrease in I _to (20.0 ± 1.6pApF^-1 vs. 28.5 ± 2.0pApF^-1, n = 15, p < 0.001, V _Pip = 40 mV) within 5 min. Control solution resulted in a significantly smaller reduction in I _to (17 ± 3 %, p < 0.001, p < 0.01 compared with MBCD). MBCD induced a 38 ± 9 % increase in the non-inactivating current component (I _sus) (10.1 ± 0.6pApF^-1 vs. 7.6 ± 0.4pApF^-1, n = 15, p < 0.001). This effect was absent in control solution. The increase in I _sus was not sensitive to 100 μM 4-aminopyridine or 20 mM tetraethylammonium, making a contribution of Kv1.5 or Kv2.1 unlikely. In conclusion, in rat ventricular cardiomyocytes, a fraction of Kv4.2 and KChIP2 is localized in lipid rafts. Membrane cholesterol depletion results in ~12 % net reduction of I _to, a redistribution of the channel proteins Kv4.2 and KChIP2 and an increased delayed rectifier current.