The constituents of the antitumor agent auromomycin have been analyzed to determine their DNA-breakage activities. Spectral analysis showed that the methanol extract contained 70% of the non-peptide chromophore, whereas the residue contained 20%. Amino acid analysis of the methanol extract showed that it contained 21%-26% of the original auromomycin polypeptides. The DNA-degradation activity of the extract was 121% +/- 28% of that of the untreated auromomycin, whereas that of the residue was only 22% +/- 3.8%. Mixing of the residue and the methanol extract resulted in the loss of three-fourths of the total activity. Agarose gel electrophoretic analysis showed that the single-strand DNA breakage activity of the methanol extract was 6.5-fold greater than that of the double-strand DNA-breakage activity. The difference in the total DNA-cleavage activity of the untreated, methanol-treated, and remixed auromomycin preparations may suggest the occurrence of certain non-peptide chromophore-polypeptide interactions in both the untreated and the remixed preparations. This is consistent with the fluorescent changes observed upon mixing of the extract and residue. Fractionation of the methanol extract by Sephadex chromatography revealed that several column fractions which were enriched with non-peptide chromophore relative to the polypeptides contained in them still had significant DNA-degradation activity. These studies suggest that the non-peptide chromophore in the auromomycin preparation may contribute to most of the observed DNA breakage activity.