The DNA binding behavior of a tricationic cyanine dye (DiSC₃₊(5)) was studied using the [Poly(dA-dT)]₂, [Poly(dI-dC)]₂ and Poly(dA)·Poly(dT) duplex sequences and the Poly(dA)·2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA)·Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]₂ duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]₂ sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J. L., et al. J. Am. Chem. Soc. 121, 2987-2995 (1999).] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.