Rho transcription termination factor mutant, F355W, showed tryptophan fluorescence intensity approximately twice that of wild-type Rho at equivalent protein concentrations and underwent a decrease in relative fluorescence intensity at 350 nm when 100 mM ATP was added in the presence or absence of RNA. Titration of this fluorescence quenching with varying concentrations of ATP (0–600 mM), where Rho is shown to exist as a hexamer (400 nM Rho), revealed tight and loose ATP binding sites. Bicyclomycin, a specific inhibitor of Rho, increased the tight ATP binding and was used to calibrate ATP-induced fluorescence quenching by using [g-32P]ATP filter binding. For the Rho mutant F355W, three tight (Kd1 = 3 ± 0.3 mM) and three loose (Kd2 = 58 ± 3 mM) ATP binding sites per hexamer were seen on Scatchard analysis in the absence of bicyclomycin and poly(C). In the presence of bicyclomycin, the Kd1 changed from 3.0 to 1.4 mM but Kd2 underwent a lesser change. The non-hydrolyzable ATP analogue, g-S-ATP, gave a similar profile with three tight (Kd1 = 0.2 mM) and three loose (Kd2 = 70 mM) ATP binding sites per hexamer. Adding poly(C) to F355W did not alter the Kd1 or Kd2 for ATP or for g-S-ATP. ADP-induced quenching produced 5.5 loose (Kd = 92 mM) binding sites in the absence of poly(C), and the binding became weaker (Kd = 175 mM) in the presence of poly(C). The data suggests that in the presence of ADP Rho has six equivalent nucleotide binding sites. When ATP was added these sites converted to three tight and three loose binding loci. We propose an alternating ATP site mechanism where ATP binding creates heterogeneity in the ATP binding in adjacent subunits, and suggest that ATP binding to a neighboring loose site stimulates hydrolysis at a neighboring tight binding site.