The interactions of the antitumor antibiotics, chromomycin A,, with a variety of metal cations in the pH range of 3.0-8.5 were systematically studied by CD, absorption, and 'H-nmr spectroscopies. Results were compared with thaw obtained in the presence of increasing amounts of calf thymus DNA. The negatively charged chromomycin A,, pK, 6.3, forms aggregates that become ordered and smaller in size, in the presence of variety of metal cations. Spectrophotometric titrations have shown that binding of the neutral drug to DNA at pH 4.5 does not require divalent cations, although the strength of the binding is greatly enhanced in their presence. At higher pH values ( > 7.0) and low DNA/drug ratio (< 20), the metal cations are necessary to induce the binding between chromomycin A, and DNA. At higher DNA/drug ratios (> 100 : I), an appreciable proportion of the drug is bound even in the absence of divalent cations. Its binding affinity to the DNA is enhanced in the presence of these cations and at low pH values. Therefore, we conclude that chromomycin A, binds to DNA in two related modes, in the presence and in the absence of divalent cations. The spectral data accumulated indicate the metal cation is involved in the binding of the drug to the DNA by forming a drug-metal-DNA ternary complex.