The association constant for the interaction of daunomycin with DNA was determined as a function of temperature (using [3H] daunomycin in conventional equilibrium dialysis cells) and ionic strength (using a spectrophotometric titration method). The association constant varied between 3.1 X 10(-6) M(-1) (4 degrees C) and 3.9 X 10(5) M(-1) (65 degrees C). The free energy change was -8.2 to -8.8 kcal/mol, the enthalpy change -5.3 kcal/mol and the entropy change +10 to +11 eu, all values being consistent with that expected of an intercalation process. The apparent number of intercalation sites detected (0.15 to 0.16 per nucleotide) was independent of temperature. The large positive entropy change accompanying the interaction appears to be due to extensive release of water from the DNA and daunomycin. The apparent number of binding sites increased dramatically with decrease of ionic strength, although the apparent association constant remained largely unaffected by ionic strength.