Fluorescence titration methods and equilibrium dialysis were used to study the thermodynamics of the interaction of doxorubicin, the β anomer of doxorubicin, daunorubicin, and hydroxyrubicin with DNA. All of these except hydroxyrubicin carry a net charge of +1 at neutral pH, arising from the protonation of the daunosamine moiety. Hydroxyrubicin is a synthetic anthracycline antibiotic in which the amine moiety has been replaced by a hydroxyl group, which is uncharged but polar at neutral pH. The comparative binding studies we describe offer a unique opportunity to evaluate the electrostatic contributions to the DNA binding free energy of these anthracycline antibiotics and to test specific predictions arising from current polyelectrolyte theory as applied to ligand-DNA interactions. We find that the quantity (δ log K/δ log [M+]) changes from a value of −0.95 for doxorubicin to −0.18 for hydroxyrubicin. The latter value is in excellent agreement with the value of −0.24 predicted by the theory of Friedman and Manning for the interaction of an uncharged intercalating ligand with DNA. The DNA binding free energy decreases from −8.8 kcal mol−1 for doxorubicin to −7.2 kcal mol−1 for hydroxyrubicin under solution conditions of 200 mM Na+, pH 7.0, 20 °C. The results we obtain allow us to dissect the DNA binding free energy into its electrostatic and nonelectrostatic components. Knowledge of these values helps to clarify the precise role of the amine group in doxorubicin binding to DNA. In addition, comparison of the DNA binding of doxorubicin with the β anomer of doxorubicin provides a striking example of stereoselective antibiotic binding to DNA. © 1993, American Chemical Society. All rights reserved.