Drug resistance is a major cause of the failure of anticancer chemotherapy. Multidrug resistance is often caused by overexpression of the P-glycoprotein (Pgp) or the multidrug resistance-related protein (MRP). In the present study, we compared daunorubicin (DNR) accumulation, subcellular distribution, and the effect of modulators on drug accumulation and subcellular distribution in the Pgp-expressing K562 cell line and the MRP-expressing HL60 cell line using reverse-transcriptase polymerase chain reaction, MTT (3-[4, 5-dimethylthiazol-z-yl]-2,5-diphenyltetrazolium bromide) drug cytotoxicity assay, fluorocytometry, and confocal laser scanning microscopy. The 2 resistant cell lines exhibit similar levels of resistance to DNR and decreased drug accumulation. Altered drug subcellular distribution in the resistant cell lines compared to that in the sensitive cell lines was shown and, moreover, differences in drug distributions between the 2 resistant cell lines were found. DNR fluorescence in the resistant HL60 cell line was distributed into punctate regions in the cytoplasm; the nucleus and other cytoplasm were almost negative. In contrast, the resistant K562 cells showed a bright fluorescent signal located in the peripheral cytoplasm and perinuclear region; the nucleus and other cytoplasmic regions showed no signal. Use of the modulator verapamil increased drug accumulation and restored the altered subcellular distribution of the drug in the 2 resistant cell lines. The Golgi apparatus inhibitor brefeldin A had similar action in the resistant HL60 line but had little effect in the resistant K562 line. Therefore, our study suggested that there were differences between the 2 resistant cell lines in the compartments sequestering DNR.