The hydration changes that accompany the DNA binding of five intercalators (ethidium, propidium, proflavine, daunomycin, and 7-aminoactinomycin D) were measured by the osmotic stress method with use of the osmolytes betaine, sucrose, and triethylene glycol. Water uptake was found to accompany complex formation for all intercalators except ethidium. The difference in the number of bound water molecules between the complex and the free reactants (Deltan(w)) was different for each intercalator. The values found for Deltan(w) were the following: propidium, +6; daunomycin, +18, proflavine, +30; and 7-aminoactinomycin D, +32. For ethidium binding to DNA a value of Deltan(w) = +0.25(+/-0.3) was found, indicating that within experimental error no water was released or taken up upon complex formation. Intercalation association constants measured in D2O were found to increase relative to values measured in H2O for all compounds except ethidium. A positive correlation between the ratio of binding constants (K-D2O/K-H2O) and Deltan(w) was found. These combined studies identify water as an important thermodynamic participant in the formation of certain intercalation complexes.