Of several phytotoxins isolated from culture filtrates of Phoma betae Frank PS-13, an incitant of leaf spot disease of sugar beet, three have been identified as aphidicolin, 3-deoxyaphidicolin and aphidicolin-17-monoacetate. Aphidicolin is a selective inhibitor of eukaryotic DNA polymerase alpha (Ikegami et al. (1978) Nature 275, 458-460). Consequently, we studied the action mechanism of 3-deoxyaphidicolin and aphidicolin-17-monoacetate. These aphidicolin analogues markedly inhibited the in vivo DNA synthesis of sea urchin embryos and HeLa cells but not RNA and protein syntheses. Only DNA polymerase alpha, not DNA polymerase beta and gamma, was inhibited by these drugs. The mode of action of these analogues on DNA polymerase alpha from the sea urchin was competitive inhibition with respect to dCTP with Ki values of 0.44 micrograms/ml for deoxyaphidicolin and 0.89 micrograms/ml for aphidicolin monoacetate, respectively. None of the other three dNTPs competed with these drugs. A similar inhibitory mode was observed using the enzyme from HeLa cells and toad oocytes. These drugs at a concentration of 2 micrograms/ml caused a delay in the cleavage of fertilized eggs of the sea urchin and decomposition before blastulation, indicating the possibility of achromosomal cleavage because of the absence of DNA synthesis. Based on the above, it is concluded that these analogues can be used as other inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha.