The diazido derivative of ethidium bromide has been synthesized as a potential photoaffinity label and shown to be at least as effective as a mitochondrial mutagen as the parent compound, with a similar mode of action. Exposure of mitochondria of Saccharomyces cerevisiae to the compound, followed by ultraviolet-irradiation, which converts it to the highly reactive dinitrene, results in its specific binding to a single component which has been tentatively identified as the smallest polypeptide (subunit 9) of the membrane-bound ATPase. An analogus reaction is also obtained with the soluble, oligomycin-sensitive ATPase complex but not with the F1-ATPase itself. The reaction with the ATPase complex can also be monitored by fluorescence enhancement and by this attribute, as well as by other criteria, diazido-ethidium bromide, ethidium bromide itself, euflavine, N,N'-dicyclohexylcarbodiimide, 2,4-dinitrophenol, and 2-azido-4-nitrophenol all appear to compete for the same, lipophilic, binding site. A mitochondrial mutation (73/1) (see Flury, U., Feldman, F., and Mahler, H.R. (1974) J. Biol. Chem. 249, 6630-6637) produces a photoaffinity product with an altered electrophoretic mobility and molecular weight.