A series of rhodium complexes containing the phenanthrenequinone diimine (phi) ligand have been prepared which bind DNA by intercalation and, upon photoactivation, promote DNA strand breaks. In this series, the ancillary, nonintercalating bipyridyl or phenanthroline ligands have been functionalized to yield complexes containing guanidinium, amido, or amino groups arranged with defined stereochemistry for site-specific interaction with the DNA bases. Lambda-1-[Rh(MGP)(2)phi](5+) (MGP = 4-(guanidylmethyl)-1,10-phenanthroline) site-specifically targets the 6-base pair sequence 5'-CATATG-3' with a binding affinity of 1 (+/-0.5) x 10(8) M-1 while Delta-1-[Rh(MGP)(2)phi](5+) displays an affinity of 5 (+/-2) x 10(7) M-1 for 5'-CATCTG-3'. Even though these two isomers target sites which differ by only a single base, binding is highly enantioselective. The specificity is derived chiefly from interactions of the pendant guanidinium groups with the DNA bases. For the racemates of 1-[Rh(GEB)(2)phi](5+) (GEB = (4-(2-guanidylethyl)-4'-methyl-2, 2'-bipyridine) and 1-[Rh(GPB)(2)phi](5+) (GPB = (4-(2-guanidylpropyl)-4'-methyl-2,2'-bipyridine), photocleavage patterns also show the strongest site of photocleavage as 5'-CATCTG-3', the target site for Delta-1-[Rh(MGP)(2)phi](5+). Moreover, consistent with the dominance of the guanidinium groups in establishing specificity, significantly enhanced photocleavage is evident for the 1-positional isomer of these complexes, where the guanidinium moieties are directed toward the DNA (above and below the ph ligand) compared to the 2-isomer, in which the guanidinium groups are directed away from the DNA. In contrast to Lambda-1-[Rh(MGP)(2)phi](5+), Lambda-1-[Rh(GEB)(2)phi](5+) shows little cleavage at 5'-CATATG-3'; this sensitivity to linker length likely depends on the mode of recognition of 5'-CATATG-3' involving sequence-dependent unwinding of the DNA site. Analogous site-specificity or isomer-specificity is not evident with the complexes which contain pendant amido or amino functionalities. Instead these complexes appear to resemble the parent, unfunctionalized [Rh(phen)(2)phi](3+) with respect to recognition. Pendant guanidinium functionalities appear to be particularly advantageous in the construction of small molecules which bind DNA with site-specificity.