The increase in fluorescence on binding of m-phenyl substituted hydroxy derivatives of Hoechst 33258 with poly-[d(A-T)], d(CGCGAATTCGCG) 2 , and with the corresponding T 4 -looped 28-mer AATT hairpin was used to monitor binding by equilibrium titrations and stopped-flow kinetics. Replacing the p-OH substituent of Hoechst 33258 (association constant K a = 5.2 x 10 8 M -1 for 28-mer hairpin) by m-OH increases the AATT site binding energy by 1.1 kcal mol -1 , K a = 3.8 x 10 9 M -1 . Addition of a second m-hydroxy group (bis-m-OH Hoechst) further strengthens binding, giving K a = 1.9 x 10 10 M -1 , and the binding energy increases by about 2.1 kcal mol -1 compared to p-OH Hoechst. The value of K a determined at equilibrium equaled that determined from the ratio of association and dissociation rate constants from stopped-flow studies. The increase in affinity of the monohydroxy Hoechst analogue (m-OH) may originate from water-mediated hydrogen bonding with the minor groove. The further increase in affinity of the bis-m-OH derivative (whose second m-OH group must be directed away from the DNA minor groove floor) may arise from a hydrogen-bonded network of water molecules. The potential to increase binding strength through relayed water molecules is proposed as an additional possible input for lead drug design at DNA targets.