We have investigated the binding of the f1 single-stranded DNA-binding protein (gene V protein) to DNA oligonucleotides and RNA synthesized in vitro. The first 16 nucleotides of the f1 gene II mRNA leader sequence were previously identified as the gene II RNA-operator; the target to which the gene V protein binds to repress gene II translation. Using a gel retardation assay, we find that the preferential binding of gene V protein to an RNA carrying the gene II RNA-operator sequence is affected by mutations which abolish gene II translational repression in vivo. In vitro, gene V protein also binds preferentially to a DNA oligonucleotide whose sequence is the DNA analog of the wild-type gene II RNA-operator. Therefore, the gene V protein recognizes the gene II mRNA operator sequence when present in either an RNA or DNA context.