The voltage-dependent Kv1.3 potassium channels mediate a variety of physiological functions in human T lymphocytes. These channels, along with their multiple regulatory components, are localized in cholesterol-enriched microdomains of plasma membrane (lipid rafts). In this study, patch-clamp technique was applied to explore the impact of the lipid-raft integrity on the Kv1.3 channel functional characteristics. T lymphoma Jurkat cells were treated for 1 h with cholesterol-binding oligosaccharide methyl-β-cyclodextrin (MβCD) in 1 or 2 mM concentration, resulting in depletion of cholesterol by 63±5 or 75±4%, respectively. Treatment with 2 mM MβCD did not affect the cells viability but retarded the cell proliferation. The latter treatment caused a depolarizing shift of the Kv1.3 channel activation and inactivation by 11 and 6 mV, respectively, and more than twofold decrease in the steady-state activity at depolarizing potentials. Altogether, these changes underlie the depolarization of membrane potential, recorded in a current-clamp mode. The effects of MβCD were concentration- and time-dependent and reversible. Both development and recovery of the MβCD effects were completed within 1-2 h. Therefore, cholesterol depletion causes significant alterations in the Kv1.3 channel function, whereas T cells possess a potential to reverse these changes.