The specificity of the interaction between R17 coat protein and its site of translational repression on R17 RNA was studied by enzymatically synthesizing 23 sequence variants of the RNA binding site and measuring their affinity to the coat protein by a nitrocellulose filter binding assay. Experiments using oligomers truncated on the 3' and 5' termini allowed precise determination of the edges of the binding domain. Several oligomers which disrupted one or more of the base pairs in the binding site failed to bind coat protein, establishing the importance of RNA secondary structure for the interaction. Substitution at two single-stranded positions with each of the common bases affected Ka very differently. In one case, Ka was reduced substantially no matter which base was substituted for an adenine. At the other position, when a uracil was substituted with a purine, Ka decreased 10-100-fold, whereas when it was substituted by a cytosine, Ka increased about 5-fold. These studies indicate that the protein and the RNA hairpin loop interact over an extensive area and that several different types of contacts form to stabilize the complex.