The luminescence of DNA-bound [Ru(phen)₂dppz]²⁺ is shown to be highly sensitive to environmental conditions such as ionic strength, temperature, and the sequence and secondary structure of the nucleic acid, although not to bulky DNA substituents in the major groove. Each enantiomer has two characteristic lifetimes with any polynucleotide and their relative amplitudes vary as a function of binding ratio. For [poly(dA-dT)]₂ as a model sequence, the longer lifetime for Δ-[Ru(phen)₂dppz]²⁺ has been assigned to canted intercalation of the complex and the shorter lifetime is ascribed to symmetric intercalation. At a fixed binding ratio, the longer lifetime amplitude increases with increasing ionic strength, without significant change in lifetimes. Increasing temperature has a similar effect, but also affects lifetimes. In general, emission is strongest with AT-rich polynucleotides and with higher-order secondary structures, with intensity increasing as single-stranded < duplex < triplex. However, sequence-context and secondary duplex structure also influence the photophysics since emission with [poly(dA)]·[poly(dT)] is significantly higher than with [poly(dA-dT)]₂ or [poly(rA)]·[poly(rU)]. The strong influence of different environmental conditions on the emission of nucleic acid-bound [Ru(phen)₂dppz]²⁺ reflects subtle heterogeneities that are inherent elements of DNA recognition by small molecules, amplified by large changes in photophysics caused by differential exposure of the dppz nitrogens to groove hydration.