Helenalin and bis (helenalinyl) malonate, sesquiterpene lactones, were shown to be cytotoxic against the growth of P-388 lymphocytic leukemia cells in culture. DNA and protein synthesis were reduced by these agents preferentially, with RNA synthesis being affected only marginally. This study focused on the identification of the enzyme target(s) responsible for the inhibition of DNA synthesis by the sesquiterpene lactones. Purine synthesis was strongly inhibited at the IMP dehydrogenase step. Suppression of IMP dehydrogenase activity and purine synthesis paralleled the DNA synthesis inhibition with respect to both dose dependence and time of incubation with drug. Deoxyribonucleoside triphosphate pools in the P-388 cells were significantly reduced by both drugs and the DNA polymerase alpha activity was only moderately inhibited by both drugs in cytoplasmic preparation. However, inhibition of a partially purified DNA polymerase alpha was of a much greater magnitude. Activity of the ribonucleotide reductase complex was reduced by more than 50% at 100 microM concentration of either drug. The drugs appeared to affect the hydrogen donor system of the reductase complex, since the activity of the ribonucleotide reductase enzyme itself was not affected but both thioredoxin and glutaredoxin were markedly inactivated by the sesquiterpene lactones. Thymidylate synthetase activity was not affected by the sesquiterpene lactones in P-388 cells. These data suggest that the inhibition of IMP dehydrogenase and the ribonucleotide reductase complex activities by helenalin and bis (helenalinyl) melonate was the primary reason for the observed inhibition of DNA synthesis, but that inhibition of DNA polymerase alpha may also play a role. The inhibition of the sensitive enzymes is likely to be related to drug alkylation of thiol active groups of the enzymes in a manner similar to the action of N-ethylmaleimide. The mode of action of helenalin and bis (helenalinyl) malonate does not appear to be similar to that of the parthenolide-type sesquiterpene lactones which contain an epoxide moiety.