In an effort to determine the in vivo targets for ethidium bromide, the promising new technique of photoaffinity labeling has been applied in the development of two photosensitive ethidium azido analogues. One of these, ethidium monoazide, 3-amino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, has been shown previously to have biological properties similar to those of the parent ethidium prior to photolytic activation. After photolysis, which results in the covalent attachment of the ethidium moiety, the monoazide demonstrates enhanced biological activity. The diazide, 3,8-diazido-5-ethyl-6-phenylphenanthridinium chloride, is much less active. Since nucleic acids are presumed to be one of the targets of ethidium, both the noncovalent and the covalent interactions of ethidium and these azides with calf thymus DNA were analyzed at several salt concentrations by using spectrophotometric and dialysis techniques. The results presented in this paper show that the noncovalent interaction of the monoazide with deoxyribonucleic acid (DNA) is essentially identical with that of the parent ethidium and is primarily intercalative in nature. The DNA interaction with the diazide, apparently a stacking interaction, is quite different as seen by the greater decrease in the apparent association constant at elevated salt concentrations. Furthermore, the covalent interaction of the monoazide with DNA formed with ~40% photolytic efficiency resembled that of the noncovalent complex which suggests that no reorientation of the noncovalently bound ligand is required for covalent attachment. These results demonstrate that the monoazido analogue of ethidium bromide may be useful in determining directly the targets responsible for biological activity. © 1981, American Chemical Society. All rights reserved.