Amrubicin, a completely synthetic 9-aminoanthracycline derivative, was previously shown to have potent antitumor activities against various human tumor xenografts. In this study, the in vitro activities of amrubicin and its major metabolite, amrubicinol, were examined using 17 human tumor cell lines. Amrubicinol was 5 to 54 times more potent than amrubicin, and as potent as doxorubicin, in inhibiting the growth of the cells following 3-day continuous drug exposure. Amrubicinol closely resembled doxorubicin in its profile of activities on the 17 human tumor cell lines. Cells were incubated with the drugs for 1 h, and the intracellular drug concentration and cell growth inhibition after 3 days were determined. Amrubicinol attained similar intracellular concentrations at lower medium concentrations compared to amrubicin, and the intracellular concentration of amrubicinol necessary to produce 50% cell growth inhibition was 3 to 8 times lower than that of amrubicin in 4 cell lines tested. Amrubicinol has a higher activity level inside the cells than does amrubicin. When cells were incubated with amrubicin for 5 h, a substantial amount of amrubicinol, more than 9% of that of amrubicin, was found in cells in 4 of the 8 cell lines tested. Amrubicinol may contribute to the in vitro growth-inhibitory effect of amrubicin on these cells. The results suggest that amrubicinol plays an important role in the in vivo antitumor effect of amrubicin as an active metabolite.