The complex bis(phenanthrenequinone diimine)(bipyridyl)rhodium(III), Rh(phi)2(bpy)3+, cleaves DNA efficiently in a sequence-neutral fashion upon photoactivation so as to provide a novel, high resolution, chemical photofootpring reagent. Photofootprinting of two crystallographically characterized DNA-binding agents, distamycin, a small natural product which binds to DNA in the minor groove, and the endonuclease EcoRI, which binds in the major groove, gave respectively a 5-7 base pair footprint for the drug at its A6 binding site and a 10-12 base pair footprint for the enzyme centered at its recognition site (5'-GAATTC-3'). Both footprints agree closely with the crystallographic results. The photocleavage reaction can be performed using either a high intensity lamp or, conveniently, a simple transilluminator box, and the photoreaction is not inhibited by moderate concentrations of reagents which are sometimes required for examining interactions of molecules with DNA. When compared with other popular footprinting agents, the rhodium complex shows a number of distinct advantages: sequence-neutrality, high resolution, ability to footprint major as well as minor groove-binding ligands, applicability in the presence of additives such as Mg2+ or glycerol, ease of handling, and a sharply footprinted pattern. Light activated footprinting reactions furthermore offer the possibility of examining DNA-binding interactions with time resolution and within the cell.