We studied the intracellular distribution of drugs within anthracycline-sensitive and -resistant cells by computer-assisted digitized video fluorescence microscopy. We found that the antitumor antibiotic, daunorubicin, distributes differently in anthracycline-sensitive and -resistant human leukemia cells (HL-60). Verapamil and other agents known to circumvent resistance in pleiotropic drug-resistant cell lines were able to change the pattern of distribution of daunorubicin in the anthracycline-resistant HL-60 cells back to the distribution found in anthracycline-sensitive HL-60 cells. To investigate the biochemical basis for this effect, we studied the distribution of daunorubicin and doxorubicin in a hydrophobic/hydrophilic (membrane/cytoplasmic) environment using the two-compartment cell-free system of Folch. Our results demonstrate that various unrelated drugs known to overcome resistance will also change the distribution of the anthracyclines in the hydrophobic/hydrophilic compartments. Our data allow the hypothesis that various unrelated agents known to circumvent resistance may act by altering the hydrophobic/hydrophilic solubility of anthracyclines in the resistant cell.