Hymenoic acid (1) is a natural compound isolated from cultures of a fungus, Hymenochaetaceae sp., and this structure was determined by spectroscopic analyses. Compound 1 is a novel sesquiterpene, trans-4-[(1'E,5'S)-5'-carboxy-1'-methyl-1'-hexenyl]cyclohexanecarboxylic acid. This compound selectively inhibited the activity of human DNA polymerase lambda (pol lambda) in vitro, and 50% inhibition was observed at a concentration of 91.7microM. Compound 1 did not influence the activities of the other seven mammalian pols (i.e., pols alpha, gamma, delta, epsilon, eta, iota, and kappa), but also showed no effect even on the activity of pol beta, which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. This compound also did not inhibit the activities of prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 1 could be a selective inhibitor of eukaryotic pol lambda. This compound had no inhibitory activities against two N-terminal truncated pol lambda, del-1 pol lambda (lacking nuclear localization signal (NLS), BRCA1 C-terminus (BRCT) domain [residues 133-575]), and del-2 pol lambda (lacking NLS, BRCT, domain and proline-rich region [residues 245-575]). The compound 1-induced inhibition of intact pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol lambda inhibitory mechanism of compound 1 is discussed.