Under approximately physiological conditions, the transcription termination factor rho from Escherichia coli is a hexamer of planar hexagonal geometry [Geiselmann, J., Yager, T. D., Gill, S. C., Calmettes, P., & von Hippel, P. H. (1992) Biochemistry (preceding paper in this issue)]. Here we describe studies that further define the quaternary structure of this hexamer. We use a combination of chemical cross-linking and treatment with mild denaturants to show that the fundamental unit within the rho hexamer is a dimer stabilized by an isologous (or pseudoisologous) bonding interface. Three identical dimers of rho interact via a second type of isologous bonding interface to yield a hexamer with C₃ or D₃ symmetry. Cross-linking and denaturation experiments definitely rule out C₆ and C₂ symmetry for the rho hexamer. Data from fluorescence quenching, lifetime, and energy transfer experiments also argue against C₂ symmetry. The simplest symmetry assignment that is not contradicted by any experimental data is D₃; thus we conclude that the rho hexamer has D₃ symmetry. We also consider the positioning of the binding sites for RNA and ATP relative to the coordinate reference frame of the D₃ hexamer. Fluorescence energy transfer data are presented and integrated with data from the literature to arrive at a self-consistent model for the quaternary structure of the rho hexamer.