4,4'-Diacetyldiphenylurea-bis(guanylhydrazone) (DDUG), an agent very effective against several animal leukemias and tumors, was found, spectrophotometrically, to interact in a biphasic manner with several natural, native and heat-denatured, and synthetic DNAs. The spectrum of DDUG was shifted towards the visible region with a hypochromic shift reaching a maximum hypochromicity at 316 mmu at a 1 : 1 molar ratio of DDUG to DNA nucleotide. Increasing molarity of DNA nucleotide resulted in a further shift towards the visible end, but with hyperchromicity rather than hypochromicity, and reaching its peak at 323 mmu. The interaction with yeast RNA was much weaker than that with DNA. 4,4'-Diacetyldiphenylurea (DDU) did not show any interaction with DNA; its monoguanylhydrazone (DDUM) showed only a hypochromic interaction. In contrast to DDUG, methylglyoxal-bis( guanylhydrazone (CH3-G), an aliphatic bisguanylhydrazone with antileukemic properties, showed only a hypochromic interaction with DNA at low ionic strength. Unlike DDUG, CH3-G was a very weak inhibitor of the DNA polymerase reaction. The hypochromic shift of the DDUG spectrum with DNA was abolished in the presence of 15 mM sodium citrate or 500 mM NaCl but not in the presence of 150 mM NaCl or 100 mM sodium acetate. The hyperchromic shift was abolished in the presence of 8 M urea. From the results obtained with different DNAs, RNA, synthetic polynucleotides and nucleotides, it appears that the total shift of the DDUG spectrum in the presence of intact DNA can not be ascribed to interaction with a single base although a greater shift occurred in the presence of G-C rich DNA.