Asymmetric rolling circle replication of the promiscuous replicon pMV158 is initiated by the plasmid-encoded RepB protein. In vitro, purified RepB protein introduces a nick within the leading strand origin of replication by a nucleophylic attack on the phosphodiester bond at the dinucleotide GpA. Some changes within and around this dinucleotide were recognized by the protein. RepB nicked and closed supercoiled pMV158 DNA, having an optimum activity at 60 degrees C. We have imitated, in vitro, a process of rolling circle replication, since RepB was able to nick (initiation) and to covalently close (termination) single-stranded oligonucleotides containing the protein cleavage sequence. Covalent DNA-protein complexes were not found, indicating that RepB has unique features among plasmid-encoded proteins involved in rolling-circle replication or conjugative mobilization.