Cholesterol is a major lipid component of the plasma membranes, and it plays an important role in lipid organization, lateral heterogeneity and dynamics of plasma membrane. Lipid rafts, cholesterol-rich membrane domains, are assumed to play an essential role in the interactions between cell membrane and cortical cytoskeleton (Nebl et al., 2002). As we have shown earlier, the activity of non-voltage-gated sodium channels in K562 human leukaemia cells is critically dependent on F-actin organization (Negulyaev et al., 2000; Shumilina et al., 2003). Our present work was aimed to investigate possible role of lipid environment in the functions of sodium channels and their coupling with cortical cytoskeleton in K562 cells. The patch-clamp method was employed to examine the effect of methyl-β-cyclodextrin (MbCD), an agent that removes membrane cholesterol, on sodium currents. Single channel currents were recorded from cell-attached and inside-out patches essentially as described earlier (Morachevskaya et al., 2007). After incubation with MbCD (2.5 or 5 mM) an activation of sodium channels in response to cytochalasin B or D was observed in membrane fragments (inside-out mode) as well as in native cells (cell-attached mode). Unitary conductance of the channels in cholesterol-depleted cells (11.6±0.1 pS, n=8) was similar to those in control cells (11.9±0.7 pS, n=7). Inside-out experiments with the use of globular actin have indicated that filament assembly on cytoplasmic membrane side causes an inactivation of sodium channels. In sum, we conclude that cholesterol depletion did not affect essentially the biophysical characteristics, sodium channel activation and inactivation coupled with F-actin rearrangement. Our electrophysiological data imply that there is no association of non-voltage-gated sodium channels with cholesterol-rich membrane microdomains.