We have used equilibrium dialysis and fluorescence and absorbance titration to study the interaction of daunomycin with DNA. Our data at 200 mM Na+ are best fit by the neighbor exclusion model, with K = 7.0 x 10(5) M-1 and an exclusion parameter of three to four base pairs. The binding is dependent on ionic strength, with d log K/d log [Na+] = -0.84, from which we may estimate quantitatively ion release and the binding free energy corrected for the free energy of counterion release. From the temperature dependence of the binding constant, we find the binding to be exothermic, with a van't Hoff enthalpy of -12.8 kcal/mol. Competition dialysis experiments show that G+C base pairs are slightly preferred as binding sites for the drug and suggest that daunomycin binds preferentially to G+C pairs at low r. Cesium chloride density gradient sedimentation experiments provide an experimental demonstration of this preference. Daunomycin increases the Tm for DNA melting by some 30 degrees C as binding approaches saturation, with biphasic melting at low drug/base pair ratios. The data from these equilibrium studies are consistent with intercalative binding of daunomycin and provide a solid foundation for further structural and kinetic studies.