Escherichia coli transcription termination factor Rho (EC 3.6.1.3) releases nascent RNA from transcription complexes in a reaction which requires ATP hydrolysis. To understand the structure of the ATPase active site, we employed an analog of ATP, 8-azidoadenosine 5'-triphosphate (8-azido-ATP) as a photoaffinity labeling agent. 8-Azido-ATP interacts nearly normally with the active site of Rho. It binds to 3 sites per Rho hexamer with a 100 microM KD and is a substrate with a Vmax 5% that of ATP and a Km of 18 microM. Under UV irradiation, 8-azido-ATP makes covalent bonds with Rho, inactivating its ATPase. Rho is protected from this inactivation by the presence of ATP. We used [alpha-32P]8-azido-ATP to label the active site and identify residues involved in ATP binding. Labeled tryptic peptides of the modified Rho were purified by Fe(3+)-iminodiacetic acid affinity chromatography and reverse-phase C18 column high performance liquid chromatography. We identified a single peptide, Gly174-Lys184, that is labeled by 8-azido-ATP and protected from labeling in the presence of ATP. The modified amino acid is Lys181, whose conservative replacement by Gln181 gives rise to a poorly active enzyme (Dombroski, A. J., Brennan, C. A., Spear, P., and Platt, T. (1988a) J. Biol. Chem. 263, 18802-18809). Lys181 probably participates in binding the phosphoryl groups of ATP. Incorporation of one 8-azido-ATP per Rho hexamer is sufficient to cause inactivation, a result that indicates that the active sites of Rho interact in RNA-dependent ATP hydrolysis.