Eukaryotic cells reportedly contain at least fourteen types of DNA polymerase (pol. α,β,γ,δ,ε,ζ,η,θ,ι,κ,λ,μ,σ and Rev1). We screened for natural compounds that selectively inhibit pol. λ. The purpose was to use the compounds as tools and molecular probes to distinguish DNA polymerases and to clarify their biological and in vivo function. There have been no previous reports of inhibitors capable of distinguishing among pol. β, and λ. Petasiphenol, a bio-antimutagen isolated from a Japanese vegetable, Petasites japonicas, selectively inhibits the activities of mammalian pol. λ in vitro. The compound did not influence the activities of replicative DNA polymerases such as α,δ and ε, but also showed no effect even on the pol. β activity, the three-dimensional structure of which is thought to be highly similar to pol λ. The inhibitory effect of petasiphenol on intact pol λ including the BRCA1 C-terminus (BRCT) domain was dose-dependent, and 50% inhibition was observed at a concentration of 7.8μM. The petasiphenol-induced inhibition of the pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. Petasiphenol did not only inhibit the activity of the truncated pol λ including the pol β-like core, in which the BRCT motif was deleted in its N-terminal region. BIAcore analysis demonstrated that petasiphenol bound selectively to the N-terminal domain of pol λ, but did not bind to the C-terminal region. We found here that another phenolic compound, curcumin (diferuloylmethane), which is structurally quite similar to petasiphenol, was also a potent pol. λ inhibitor. The IC_<50> values of curcumin was 7.0μM. Based on these results, the pol λ-inhibitory mechanism of phenolic compounds such as curcumin is discussed.