Emission of Delta-Ru(phen)(2)dppz(2+) bound to nucleic acid polymers of different sequence has been investigated by time-resolved luminescence spectroscopy and the effect of major and minor groove DNA binding agents on the luminescence profile of the complex evaluated. In the presence of a 1:1 mixture of poly d(AT) and poly d(GC), the excited-state decay of Delta-Ru(phen)(2)dppz(2+) can be described by a linear combination of the decay profiles in the presence of poly d(AT) and poly d(GC) independently. This analysis indicates that similar to 85% of the complexes are bound to poly d(AT) and that the metallointercalator preferentially occupies AT sites in mixed-sequence polymers such as calf thymus or T4 DNA. When rac-Ru(phen)(2)dppz(2+) bound to [d(5'-GAGTGCACTC-3')(2)] is titrated with the major groove intercalator Delta-alpha-[Rh[(R,R)-Me(2)trien]phi](3+), the ruthenium emission yield decreases while the absorbance of the pi-pi* transition centered on the dppz ligand increases, until saturation behavior is observed at a 1:1 Rh/duplex ratio. These titrations indicate that Ru(phen)(2)dppz(2+) is displaced from the major groove by the rhodium complex. In contrast, for rac-Ru(phen)(2)dppz(2+) bound to poly d(AT), addition of the minor groove binding agent distamycin produces an increase in ruthenium emission which saturates at similar to 1 distamycin/5 bp, consistent with the double helix being able to accommodate major and minor groove binders simultaneously. Distamycin has no effect on the emission of Ru(phen)(2)dppz(2+) emission bound to poly d(GC). These photophysical studies establish a sequence preference in binding to DNA by Ru(phen)(2)dppz(2+) as well as providing support for the original assignment by NMR of ruthenium intercalation from the major groove side of the DNA helix.