Previous studies have shown that the single-stranded DNA binding protein of bacteriophage f1 (gene V protein) represses the translation of the mRNA of the phage-encoded replication protein (gene II protein). We have characterized phage mutations in the repressor and in its target. Using a gene II-lacZ translational fusion, we have defined a 16-nucleotide-long region in the gene II mRNA sequence that is required in vivo for repression by the gene V protein. We have shown that in vitro the binding affinity of the gene V protein is at least 10-fold higher to an RNA carrying this sequence than to an RNA lacking it. We propose that this sequence constitutes the gene II mRNA operator.