The binding of C₁₀ RNA oligomers to wild type and mutant Escherichia coli transcription termination factor Rho provides a model for the enzyme-RNA interactions that lead to transcription termination. One surprising finding is that wild type Rho binds between five and six C₁₀ oligomers per hexamer with K_D = 0.3 M, and five to six additional C₁₀ molecules with K_D = 7 M. Previously, approximately half this number of oligomer binding sites was reported (Wang, Y. and von Hippel, P. H. (1993) J. Biol. Chem., 268, 13947-13955); however, the E155K mutant form of Rho, thought at the time to be wild type, was used in that work. Present results with E155K Rho agree with the earlier work. C₁₀ binding with mutant forms of Rho that are altered in RNA interactions, bearing amino acid changes F62S, G99V, F232C, T286A, or K352E, indicate that the higher affinity binding sites constitute what has been termed the primary RNA site, and the lower affinity sites constitute the secondary sites. The binding data together with the crystal structures for wild type Rho (Skordalakes, E. and Berger, J. M. (2003) Cell 114, 135-146) support structurally distinct locations on Rho for the two classes of C₁₀ binding sites. The results are consistent with participation of residues 33 Å apart in secondary site RNA interactions. The data further indicate that not all RNA sites on Rho must be filled for full ATPase and transcription termination activity, and suggest a model in which RNA binding to the higher affinity sites leads to a protein conformation change that exposes the previously hidden lower affinity sites.