Isothermal titration calorimetry, UV melting, and competition dialysis techniques have been used to examine the binding of isomeric 1,4- and 2,6-bis(omega-aminopropionamido)-substituted anthracene-9,10-diones (anthraquinones) with dA(n) . dT(n) duplexes and dT(n)-dA(n) . dT(n) triplexes. Recent footprinting studies [Fox, K. R.; Polucci, P.; Jenkins, T. C.; Neidle, S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7887-7891] indicate that 2,6 derivatives, but not their 1,4 counterparts, differentially stabilize triple-stranded DNA and may have application in antigene chemotherapy. Thermodynamic investigations are here reported for interaction with dA(18). dT(18) and dT(18)-dA(18). dT(18). The 2,6 compound shows preferential tripler binding, with K-b values of 1.8 x 10(4) M (duplex)(-1) and 2.2 x 10(5) M (tripler)(-1) at 25 degrees C in aqueous solution, pH 6.0, whereas the 1,4 isomer favors duplex binding, with K-b values of 1:1 x 10(5) M (duplex)(-1) and 3.5 x 10(4) M (tripler)(-1). Binding to the preferred DNA is enthalpically driven for each ligand, whereas binding to the disfavored DNA is either entropically driven or enthalpy/entropy compensated. Further, the binding site sizes (3.6 base pairs/base triplets) suggest DNA intercalation. Competition dialysis studies with poly(dA). poly(dT) and poly(dA). poly(dT)(2) confirm these binding preferences, and qualitative support is provided from UV melting experiments. Such studies reveal tripler disruption by the 1,4 isomer at low drug concentrations while the 2,6 compound effects stabilization toward thermal tripler denaturation. Spectrophotometric studies of each free ligand indicate self-association in aqueous solution; with dimerization constants at 25 degrees C of (2.9 +/- 0.2) x 10(3) and (3.2 +/- 0.1) x 10(3) M(-1) respectively for the 1,4 and 2,6 isomers. Taken together, these data provide a firm thermodynamic basis for the contrasting duplex/tripler binding preferences of this isomeric family of ligands.