The DNA binding free energy of eight anthracycline antibiotics was determined as a function of NaCl concentration. Compounds were chosen for study that differed from the parent compounds, doxorubicin or daunorubicin, at a single chemical substituent. Determination of the salt concentration dependence of the binding constant allowed us to dissect the DNA binding free energy of each compound into its component nonelectrostatic and polyelectrolyte contributions. Comparison of the nonelectrostatic free energy contribution allowed us to evaluate the net energetic contribution of specific functional groups to DNA binding. These quantitative data revealed a surprisingly large and favorable energetic contribution (2 kcal (mol-1)) of the groove-binding daunosamine moiety and a substantial energetic penalty for alteration of its stereochemistry. The energetic cost of removal of hydroxyl groups at the C-9 and C-14 positions (which structural studies indicate may participate in hydrogen-bonding interactions with the DNA) was approximately 1 kcal mol(-1). Replacement of the 3'-amino group with a hydroxyl group led to a loss of 0.7 kcal mol(-1) in binding free energy, above and beyond the energetic penalty resulting from the removal of its positive charge from the antibiotic. The results and analysis presented here provide a rigorous and detailed description of structure-DNA affinity relationships among anthracycline antibiotics. The results are of general interest in understanding how total ligand binding free energies are partitioned among substituents and will be useful in the formulation of rules for the rational design of novel DNA binding agents.