Cloning of specific regions of plasmid RSF1010, in conjunction with in vitro replication studies, has revealed three novel genes: repA, repB, and repC. They are clustered in one region of the plasmid, separated from the origin of replication by regions that are not essential for plasmid viability in an Escherichia coli host. In vivo, a 2.1-kilobase segment of the plasmid, bearing the replication origin, can establish itself as an autonomous replicon if the DNA region carrying the three rep genes is present in the same cell on an independent plasmid. In vitro, RSF1010 DNA is efficiently replicated by an ammonium sulfate fraction from the E. coli extract, provided the extracts are prepared from cells that can supply the required rep gene products. Using cells containing the cloned rep gene region as a source of elevated levels of the rep proteins, we have partially purified these proteins in functional form. When added to an enzyme fraction derived from plasmid-free cells, they specifically promote the replication of plasmid DNA bearing the RSF1010 origin.