Quinone antibiotics, alpha- and beta-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 alpha- and beta-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 alpha-rubromycin against nucleotide substrate were 0.66 and 0.17 microM for DNA polymerase alpha and beta (pol. alpha and beta), respectively, and those of beta-rubromycin was 2.40 and 10.5 microM, respectively. Alpha-rubromycin strongly inhibited the pol. beta activity, and showed the strongest pol. beta inhibitory effect reported to date. At least on pol. beta, alpha-rubromycin was suggested to bind to the active region competing with the nucleotide substrate, and subsequently inhibit the catalytic activity. alpha-Rubromycin directly competed with the nucleotide substrate, and indirectly but simultaneously and non-competitively disturbed the template-DNA interaction with pol. beta.