The cellular accumulation and disposition of the anthracycline antitumor antibiotic aclacinomycin A (ACM) were compared to those of daunorubicin. Although both drugs were avidly accumulated by cells, intracellular concentrations of ACM were two to three times those of daunorubicin. Whereas lowered temperature (0°) reduced intracellular accumulation of both drugs, 10 mm sodium azide had no effect on accumulation of either ACM or daunorubicin. Both drugs exited from cells placed in drug-free medium, a process that was reduced at 0° but not altered by 10 mm sodium azide. Unlike whole cells, isolated nuclei accumulated more daunorubicin than ACM. This process was not altered at 0°. Both drugs were lost from nuclei placed in drug-free buffer, a process that was reduced at 0°. Unlike daunorubicin, which localized in cell nuclei, ACM localized in the cytoplasm with no detectable nuclear fluorescence. Although both drugs produced dose-dependent inhibitions of [3H]thymidine and [3H]uridine incorporation by L1210 and P388 cells, ACM inhibited both processes at lower concentrations than did daunorubicin. While daunorubicin inhibited [3H]thymidine incorporation more effectively than [3H]uridine incorporation, the reverse was observed with ACM.