Cholesterol is the most abundant lipid component of mammalian cells and it plays a crucial role in organization, lateral heterogeneity and dynamics of plasma membrane. Particularly, the level of cellular cholesterol determines functional compartmentalization of membrane lipids and proteins into ordered microdomains (lipid rafts), which may serve as scaffolds for signaling complexes. Cholesterol-enriched rafts are assumed to be implicated in various cellular reactions involving actin cytoskeleton rearrangements. Changes in cholesterol metabolism may affect membrane-cytoskeleton interactions modulating key functions in living cells. Lipid raft-associated signaling was shown to be crucial for development of a number of pathological processes, including malignant transformation. Nowadays, known cholesterol-lowering agents of statin family targeting rate-limiting step of mevalonate synthetic pathway are considered as potential instruments in anticancer therapy. The present study focuses on the physiological mechanisms of action of cholesterol-targeting drugs on membrane organization coupled with cytoskeleton in transformed cells. We compared the effects of selective sterol acceptor methyl-beta-cyclodextrin (MbCD) and HMG-CoA-reductase inhibitor simvastatin in transformed fibroblasts 3T3B-SV40. The depletion of membrane cholesterol by MbCD resulted in a disruption of lipid rafts in plasma membrane as it was revealed by fluorescent labelling of marker ganglioside GM1. Alterations of lipid order of cell membranes after MbCD treatment have been confirmed by probing with lipid-sensitive dye di-4-ANEPPDHQ. In contrast, incubation of the cells with simvastatin caused no changes in raft integrity in plasma membrane. Cholesterol-depleting treatment with MbCD induced actin assembly and intensive stress fiber formation in transformed fibroblasts whereas simvastatin effects on cytoskeleton were principally different. Rearrangements of actin structures after simvastatin application are likely to be due to the inhibition of isoprenoid synthesis rather than alterations in the level of membrane cholesterol. Collectively, our data are consistent with the notion that the disruption of lipid rafts is the upstream triggering mechanism in cholesterol-dependent filament assembly in transformed cells. Importantly, we show that the inhibition of Src-kinases did not prevent MbCD-induced actin polymerization and stress fiber formation in 3T3B-SV40 fibroblasts. These results indicate that Src-kinase-independent membrane mechanisms are likely to be involved in cholesterol-regulated cytoskeleton reorganizations coupled with reversion of transformed phenotype.