Replication of mini-F plasmid in Escherichia coli requires the plasmid-encoded RepE initiator protein and a number of host factors and is regulated by interaction of RepE with specific sequences near the replication origin, ori2. We have examined DNA binding properties of several hyperactive mutant RepE proteins with single amino acid substitutions. Plasmids carrying these (repE) mutations, unlike the parental plasmid, can replicate in bacterial hosts lacking the heat shock sigma factor (sigma 32) or deficient in the DnaK, DnaJ, or GrpE heat shock protein. Using gel-retardation assays, the mutant RepE proteins were shown to bind the ori2 repeated sequences with much increased affinities compared to the wild type RepE, whereas they bound to the repE operator with slightly reduced affinities. These results agreed well with the properties of mutant RepE proteins studied in vivo and accounted for the high RepE initiator activities and the high copy numbers of mutant plasmids. In addition, the DnaJ heat shock protein was found to markedly enhance the binding of wild type RepE to ori2 or the operator. DnaK protein with or without ATP failed to show such enhancements. Thus, among the heat shock proteins required for mini-F replication, DnaJ appears to play a major role in RepE binding to ori2 and the operator, perhaps accompanied by RepE activation.