The region responsible for replication of the megaplasmid pAtC58 in the nopaline-type Agrobacterium tumefaciens strain C58 was determined. A derivative ofa Co1E1 vector, pBluscript SK-, incapable of autonomous replication in Agrobacterium spp, was cloned with a 7.6-kb Bg1II-HindIII fragment from a cosmid clone of pAtC58, which contains a region adjacent to the operon for the utilization of deoxyfructosyl glutamine (DFG). The resulting plasmid conferred resistance to carbenicillin on the A. tumefaciens strain UIA5 that is a plasmidfree derivative of C58. The plasmid was stably maintained in the strain even after consecutive cultures for generations. Analysis of nested deletions of the 7.6-kb fragment showed that a 4.3-kb BglII-XhoI region sufficiently confers replication of the derivative of the ColE1 vector on UIA5. The region comprises three ORFs, which have high homologies with repA, repB, and repC of plasm ids in virulent Agrobacterium spp. including pTiC58, pTiB6S3, pTi-SAKURA, and pRiA4b as well as those of symbiotic plasmids from Rhizobium spp. Phylogenie analysis showed that rep genes in pAtC58 are more closely related to those in pRiA4 than to pTi plasmids including pTiC58, suggesting that the two inborn plasmids, pTiC58 and pAtC58, harbored in C58 evolved from distinct origins.