Repair polymerases participating in unscheduled DNA synthesis in isolated liver nuclei, bleomycin-treated permeable cells and in ultraviolet-irradiated living cells were studied using two specific inhibitors of DNA polymerases, aphidicolin and 2', 3'-dideoxythymidine-5'-triphosphate. Unscheduled, i.e., repair, DNA synthesis in rat liver nuclei, and in bleomycin-treated permeable SR-C3H/He and XC cells was mostly attributed to DNA polymerase beta. Unscheduled DNA synthesis in human liver nuclei, bleomycin-treated permeable HeLa and HEp-2 cells, and in ultraviolet-irradiated HeLa, HEp-2 and XC cells was partially inhibited by the polymerase alpha-specific inhibitor, aphidicolin. The results suggested that both DNA polymerase alpha and beta participated in unscheduled DNA synthesis, though the respective degrees of participation differed depending on cell type and the nature and degree of DNA damage.