UV-vis and CD spectroscopy reveal that a tricationic cyanine dye spontaneously assembles into a helical J-aggregate in the presence of a double-helical DNA template. The stability of the J-aggregate is strongly dependent on the dye concentration and DNA length in a manner that reflects a high degree of cooperativity in formation of the aggregate. Slight changes in environmental conditions such as temperature and ionic strength result in interconversion between J- and H-aggregates. The aggregate likely consists of dimeric units assembled in an offset, face-to-face orientation within the minor groove of the DNA template, analogous to an earlier report of I-I-aggregation on DNA by a related cyanine dye. A model is proposed that relates the two aggregate structures by translation of one monomer from a given dimer along the floor of the minor groove. This translation requires adjacent monomers to also translate, leading to the observed cooperativity.