Quinone antibiotics, α- and β-rubromycin, were originally found as inhibitors of retroviral reverse transcriptase. We investigated the effects of these agents on DNA metabolic enzymes including DNA and RNA polymerases as retroviral reverse transcriptase is a kind of the polymerase. As expected, we found that α- and β-rubromycin strongly inhibited not only the retroviral reverse transcriptase activity, but the activities of the mammalian DNA polymerases, telomerase and terminal deoxynucleotidyl transferase in vitro. These agents should therefore be classified as DNA polymerase inhibitors. The Ki values of α-rubromycin against nucleotide substrate were 0.66 and 0.17 μM for DNA polymerase α and β (pol. α and β), respectively, and those of β-rubromycin was 2.40 and 10.5 μM, respectively. α-Rubromycin strongly inhibited the pol. β activity, and showed the strongest pol. β inhibitory effect reported to date. At least on pol. β, α-rubromycin was suggested to bind to the active region competing with the nucleotide substrate, and subsequently inhibit the catalytic activity. α-Rubromycin directly competed with the nucleotide substrate, and indirectly but simultaneously and non-competitively disturbed the template-DNA interaction with pol. β.