The repAC operon of plasmid RSF1010 consists of the genes for proteins E, F, RepA (DNA helicase), and RepC (origin-binding initiator protein) and is transcriptionally initiated by a promoter called P4. We have studied the expression of the repAC operon in vivo by using fusions to the lacZ reporter gene. The results show that the product of the second gene, F, autoregulates the operon by inhibiting transcription from P4. To verify its properties postulated from the in vivo studies and to initiate its biochemical characterization, we have purified the F protein from an overproducing E.coli strain constructed in vitro. Purification was based on a gel retardation assay for detection of P4-specific DNA binding. Subsequent DNase footprinting of the F binding sites showed clear protection around two partially symmetric P4 sequences of 16 bp, each of which matches the symmetric consensus sequence, GCGTGAGTACTCACGC, in at least 13 positions. The native repressor, as judged from gel filtration, velocity sedimentation and crosslinking studies, exists as a dimer in dilute solution; its monomeric subunit, as predicted from DNA sequence and N-terminal protein sequence data, consists of 68 amino acids and has a calculated M tau = 7,673.