The Escherichia coli protein DnaA and the plasmid RK2-encoded TrfA protein are required for initiation of replication of the broad host range plasmid RK2. The TrfA protein has been shown to bind to five 17-base pair repeat sequences, referred to as iterons, at the minimal replication origin (oriV). Using DNase I footprinting and a gel mobility shift assay, purified DnaA protein was found to bind to four DnaA consensus binding sequences immediately upstream of the five iterons at the RK2 origin of replication. Binding of the TrfA protein to the iterons results in localized strand opening within the A+T-rich region of the replication origin as determined by reactivity of the top and bottom strands to potassium permanganate (KMnO4). The presence of either the E. coli DnaA or HU protein is required for the TrfA-mediated strand opening. Although the DnaA protein itself did not produce an RK2 open complex, it did enhance and/or stabilize the TrfA-induced strand opening.