Contributions of hydroxyethyl functions to the DNA binding affinities of substituted anthracenes are evaluated by calorimetry and spectroscopy. Isothermal titration calorimetry indicated that binding of the ligands to calf thymus DNA (5 mM Tris buffer, 50 mM NaCl, pH 7.2, 25°C) is exothermic. The binding constants increased from 1.5 × 104 to 1.7 × 106 M-1 as a function of increase in the number of hydroxyethyl functions (0-4). DNA binding was accompanied by red-shifted absorption (∼630 cm-1), strong hypochromism (>65%), positive induced-circular dichroism bands, and negative linear dichroism signals. DNA binding, in general, increased the helix stabilities to a significant extent (ΔTm ≈7°C, ΔΔH ≈ 3 kcal/mol, ΔΔS ≈ 6-20 cal/K-mol). The binding constants showed a strong correlation with the number of hydroxyethyl groups present on the: anthracene ring system. Analysis of the binding data using the hydrophobicity parameter (Log P) showed a poor correlation between the binding affinity and hydrophobicity. This observation was also supported by a comparison of the affinities of probes carrying N-ethyl (Kb = 0.8 × 10 5 M-1) versus N-hydroxyethyl side chains (Kb = 5.5 × 105 M-1). These are the very first examples of a strong quantitative correlation between the DNA binding affinity of a probe and the number of hydroxyethyl groups present on the probe. These quantitative findings are useful in the rational design of new ligands for high-affinity binding to DNA.