The function of transcription termination factor rho from Escherichia coli is dependent upon its ability to bind RNA. To delineate the extent of the RNA-binding domain in the rho polypeptide, plasmid-borne copies of altered forms of the rho gene were expressed to yield truncated versions. These proteins were then isolated and assayed for their ability to bind an RNA oligonucleotide [oligo(C)8] using an ultraviolet light-induced cross-linking assay. A fragment consisting of the first 116 amino acid residues, rho(1-116), bound oligo(C)8 with nearly the same affinity and specificity as the intact protein. Smaller derivatives lacking 5, 13, or 22 residues from the N terminus or with 2 fewer residues at the C terminus bound RNA with reduced affinity, while derivatives lacking 27 N-terminal residues or having just the first 109 residues were unable to bind RNA. Derivatives lacking N-terminal residues were considerably less soluble than rho(1-116). The physical properties of rho(1-116) indicate that it possesses approximately 20% each of alpha-helix and beta-sheet and is monomeric in solution. Thus, the results show that this fragment, which contains an RNP1 sequence motif, will be a good model for future physical-chemical studies of the protein-RNA interactions of rho.