As described previously (Mizushina Y., Tanaka N., Yagi H., Kurosawa T., Onoue M., Seto H., Horie T., Aoyagi N., Yamaoka M., Matsukage A., Yoshida S., and Sakaguchi K., Biochim. Biophys. Acta, 1308, 256-262, 1996), linoleic acid (LA) inhibits the activities of mammalian DNA polymerases. We found a natural product from a basidiomycete, Ganoderma lucidum, that enhances this effect of LA in a special manner. The structure was identified to be an ergosterol peroxide, 5, 8-epidioxy-5α, 8α-ergosta-6, 22E-dien-3β-ol by spectroscopic analyses. The ergosterol peroxide (EPO) itself scarcely inhibited the activities of calf thymus DNA polymerase α(pol. α) or rat DNA polymerase β(pol. β). However, when EPO at 0.25 mM was present, 10 μM or less of LA almost completely inhibited the pol. β activity, while almost complete inhibition by LA itself was acheived at 80 μM or higher. Interestingly, under the same conditions, EPO did not affect the LA-effect on pol. α. The action mode of the EPO was discussed.