Despite recent improvements in multimodal therapies for osteosarcoma (OS) and Ewing's family of tumors (EFTs), the prognosis of relapsed cases remains very poor because of the resistance to chemotherapy. Histone deacetylase inhibitors (HDACIs), including members of the cyclic tetrapeptide family such as FK228 and apicidin, are novel antitumor agents that can induce cell cycle arrest and apoptosis in various cancer cells. HDACIs also exhibit potent antitumor effects on OS and EFTs. However, to date there have been no studies to our knowledge reporting the effects of HDACIs on drug‐resistant OS and EFTs. Here, we demonstrated that FK228 and apicidin exhibited strong resistance in doxorubicin‐resistant clones of OS and EFTs expressing P‐glycoprotein (P‐gp) and multidrug resistance‐associated protein 1 (MRP1) and that P‐gp and MRP1 might play a crucial role in the resistance mechanism to FK228 and apicidin. A P‐gp inhibitor (verapamil) and an MRP1 inhibitor (MK571) could independently reverse the resistance to FK228 and apicidin in the drug‐resistant clones. Moreover, the combination of verapamil and MK571 could enhance HDACI‐induced cell number reduction in drug‐resistant clones to a similar extent as that in their parental clones. Although these findings suggest the difficulty in treating drug‐resistant tumors expressing P‐gp and/or MRP1 with these HDACIs, the combination of P‐gp and MRP1 inhibitors might reverse the resistance to the HDACIs in the treatment of those tumors. Because HDACIs are potent and promising antitumor drugs and seem to be close to clinical use, it is necessary to pay attention to the resistance mechanisms against HDACIs. © 2005 Wiley‐Liss, Inc.