The mechanism for uptake, storage and release of daunorubicin have been studied in cultured fibroblasts. Analysis by high performance liquid chromatography of cells incubated with daunorubicin revealed that the major part of the accumulated drug did not undergo metabolic transformation. Small amounts of daunorubicinol and aglycone were formed. [ 3H]-daunorubicin was used to study membrane fluxes of the drug under different conditions. Metabolic inhibitors enhanced the influx of [ 3H]-dauno-rubicin and, under certain conditions, also reduced its efflux, indicating that the cells have an active mechanism for the outward transport of the drug. The very high intracellular drug accumulation is due to trapping in nuclei and lysosomes. Cell fractionation techniques have been used to study drug trapping under various conditions. Nuclear storage of daunorubicin is probably due to binding to DNA. Metabolic inhibitors, as well as lowering the incubation temperature, reduced the lysosomal trapping, supporting the hypothesis that the low pH in these organelles is maintained by a proton pump and that the drug is trapped in the protonated form. A hypothesis is presented, which by combining the mechanisms for membrane transport and intracellular storage of daunorubicin, gives also an explanation for the observed differences in the cellular accumulation and subcellular distribution of daunorubicin and its 14-hydroxy derivative, doxorubicin. Daunorubicin is more lipophilic than doxorubicin and will therefore diffuse faster through the cell membrane. Assuming that both substances have the same affinity for the proposed active outward transport mechanism, this will lead to a higher steady-state level of daunorubicin in the cytosol and as a consequence to a higher lysosomal storage level if the drug in the lysosomes is in equilibrium with that in the cytosol. The similarity in nuclear storage capacity for the two substances can be explained by saturation of the available storage sites.