Neocarzinostatin solubilizes chromatin from HeLa S3 nuclei by introducing strand scissions in linker regions. Multimeric nucleosome patterns are seen on both native and denaturing gel analysis. The mechanism of drug action differs from the type of chromatin digestion seen with micrococcal enzyme in that DNA damage occurs through single-strand breaks and less acid-soluble material is produced. In addition, drug-induced release of soluble chromatin from the nuclei is not very dependent upon the addition of EDTA. The monomer repeat size is larger than that found for micrococcal enzyme and contains linker regions that are partially single-stranded. Core histone proteins as well as histone H1 do not appear to be altered by drug action, although there is clear evidence that DNA damage can occur in nucleosome cores. The chromophore portion of the drug degrades chromatin as effectively as the holoantibiotic.