The Escherichia coli transcription termination factor Rho is structurally and functionally homologous to hexameric helicases that assemble into ring structures. Using stopped-flow fluorescence and presteady-state ATPase kinetics, we have determined the kinetic pathway of poly(C) RNA binding to Rho hexamer, both in the presence and in absence of ATP. These studies indicate a four-step sequential mechanism of RNA binding and reveal the respective roles of the primary and secondary RNA binding sites in initiation and ATPase activation of Rho. The primary RNA binding sites of Rho hexamer interact with poly(C) RNA at a diffusion-limited rate constant close to 8 x 10(8) m(-1) s(-1), resulting in the Rho-RNA species PR1, which subsequently isomerizes to PR2 with a rate constant 21 s(-1). The PR2 isomerizes to PR3 with a rate constant of 32 s(-1) in the presence of ATP, and the formation of PR4 from PR3 results in a species that is fully competent in hydrolyzing ATP at the RNA-stimulated rate. The PR3 to PR4 isomerization occurs at a relatively slow rate of 4.1 s(-1); thus, the presteady-state ATPase kinetics show a distinct lag due to the slow initiation step. The interactions of the RNA with the primary sites trigger ring opening, and we propose that during the last two steps, the RNA migrates into the central channel and interacts with the secondary sites, resulting in the activation of the ATPase activity. The primary RNA binding sites, in addition to promoting sequence specific initiation, kinetically facilitate loading of the RNA into the secondary sites, which are relatively inaccessible, since they are present in the central channel. These studies reveal common features used by hexameric helicases to bind nucleic acids in an efficient and specific manner.