Neocarzinostatin (NCS) is an antineoplastic antibiotic causing DNA cleavage. Actinomycin D (ActD) is an antineoplastic antibiotic, which does not cleave DNA strands. We examined the mechanism of ActD-mediated amplification of NCS-induced DNA cleavage using 32P-labeled DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene. NCS plus glutathione caused DNA cleavage at thymine and adenine residues in the absence of ActD. The addition of ActD enhanced the DNA cleavage at the sites at which the lesions on opposite strands are staggered 2 bases in a 3' direction, particularly at the 5'TCT-3'.3'-AGA-5', 5'-TGT-3'.3'-ACA-5' and 5'-ACT-3'.3'-TGA-5' sequences, suggesting that ActD amplified double-stranded DNA cleavage. The mechanism of ActD-mediated amplification of NCS-induced DNA cleavage can be explained by assuming that binding of ActD to DNA changes the DNA conformation to allow NCS to bind to DNA at the specific sequences.