The binding ofa netropsin analogue to random sequence DNA, monitored by CD, is seen dependent on the concentration ofneutral solutes. The binding free energy decreases linearly with solute osmolal concentration and the magnitude of the effect is insensitive to the chemical identity ofthe solute for betaine, sorbitol, and triethylene glycol. These solutes appear to modulate binding through their efect on water activity and changes in the hydration of the drug and DNA in the complex reaction, not through a direct interaction with the reactants or the product. The dependence of binding constant on solute concentration can be interpreted as an additional binding of some 50-60 extra solute excluding water molecules by the complex. A water sensitivity ofdrug binding is further seen from the dependence of binding constants on the type of anion in solution. Anions in the Hofmeister series strongly affect bulk water free energies and entropies. The diflerences in netropsin analogue binding to DNA with CI-, F ~, and CIO; are consistent with the effect observed with neutral solutes. The ability to measure changes in water binding associated with a specific DNA interaction is ajrst step toward correlating changes in hydration with the strength and specificity ofbinding. 0 I995 John Wiley & Sons, Inc.