Escherichia coli transcription termination factor rho is an RNA-dependent ATPase, and ATPase activity is required for all its functions. We have characterized the binding of ATP to the physiologically relevant hexameric association state of rho in the absence of RNA and have shown that there are six ATP binding sites per rho hexamer. This stoichiometry has been verified by a number of different techniques, including ultracentrifugation, ultrafiltration, and fluorescence titration studies. We have also shown that ATP can bind to isolated monomers of rho when the hexamer is dissociated with the mild denaturant myristyltrimethylammonium bromide, demonstrating that each promoter of rho carries an ATP binding site. The six binding sites that we observe in the rho hexamer are not equivalent; the hexamer contains three strong (Ka approximately 3 x 10(6) M-1) and three weak (Ka approximately 10(5) M-1) binding sites for ATP. The binding constant of the weak binding site is just the reciprocal of the enzymatic Km for ATP as a substrate; thus these weak sites, as well as the strong sites, can, in principle, take part in the catalytic cycle. The asymmetry induced (or manifested) by ATP binding reduces the symmetry of the rho hexamer from a D3 to a pseudo-D3 state. This "breakage" of symmetry has implications for the molecular mechanism of rho, because an asymmetric structure can lead to directional helicase activity by invoking directionally distinct RNA binding and release reactions (see Geiselmann, J., Yager, T.D., & von Hippel, P.H., 1992c, Protein Sci. 1, 861-873).