hsp90 facilitates acquired drug resistance of tumor cells through cholesterol modulation however independent of tumor progression acquired multidrug resistance of cancer cells challenges the chemotherapeutic interventions to understand the role of molecular chaperone hsp90 in drug adapted tumor cells we have used in vitro drug adapted epidermoid tumor cells as a model system we found that chemotherapeutic drug adaptation of tumor cells is mediated by induced activities of both hsp90 and pglycoprotein pgp although the highaffinity conformation of hsp90 has correlated with the enhanced drug efflux activity we did not observe a direct interaction between pgp and hsp90 the enrichment of pgp and hsp90 at the cholesterolrich membrane microdomains is found obligatory for enhanced drug efflux activity since inhibition of cholesterol biosynthesis is not interfering with the drug efflux activity it is presumed that the net cholesterol redistribution mediated by hsp90 regulates the enhanced drug efflux activity our in vitro cholesterol and hsp90 interaction studies have furthered our presumption that hsp90 facilitates cholesterol redistribution the drug adapted cells though exhibited antiproliferative and antitumor effects in response to 17aag treatment drug treatment has also enhanced the drug efflux activity our findings suggest that drug efflux activity and metastatic potential of tumor cells are independently regulated by hsp90 by distinct mechanisms we expose the limitations imposed by hsp90 inhibitors against multidrug resistant tumor cells