Inhibitors of DNA polymerase alpha (aphidicolin, phosphonoacetic acid, phosphonoformic acid) efficiently inhibit initiator-induced amplification of SV40 DNA sequences in the SV40-transformed Chinese hamster cell line CO631. Amplification is also inhibited by various protease inhibitors (antipain, leupeptin, aprotinin, alpha-I-antitrypsin, epsilon-amino-caproic acid, soy-bean protease inhibitor), by the non-initiating but DNA-damaging agent caffeine, and by sodium butyrate, which inhibits DNA synthesis by histone modification. In contrast, an inhibitor of topoisomerase II, nalidixic acid, enhances amplification when applied simultaneously with initiating treatment. This latter compound does not induce amplification when applied without initiator. Cycloheximide induces DNA amplification in the same way as chemical and physical carcinogens. This amplification can still be observed when protein synthesis is completely blocked. The data suggest a complex mechanism of selective DNA amplification. The possible involvement of proteases leading to a functional modification of DNA polymerase alpha is discussed.