Two regions of the nopaline-type Ti plasmid pTiC58 are important for conjugal transfer of this element to recipient bacteria. These two regions were cloned into two independent replicons to produce a binary transfer system. For one region, oriT/tra, we constructed two derivatives, pFRtra and pDCtra-5. Each contains the oriT site and the two flanking, divergently transcribed tra operons that encode the DNA processing functions associated with the relaxosome. These two plasmids also carry traR, which encodes the transcriptional activator necessary for expression of transfer genes. The two plasmids differ by the amounts of traB sequence or sequence downstream of traG present in the construct. The second replicon, pPLE2, carries the traI/trb region. The traI gene confers production of the Agrobacterium tumefaciens N-acyl homoserine lactone autoinducer, while the remaining genes in the trb operon encode components of the mating bridge. Donors harboring the two plasmids mobilized the transfer of the plasmid carrying the oriT/tra region to an A. tumefaciens recipient at frequencies similar to that at which the intact Ti plasmid transferred. Plasmid pFRtra, which encodes most of traB, was mobilized at a frequency almost 10-fold higher than was pDCtra-5, which lacks most of the gene. A. tumefaciens donors also mobilized pFRtra to Escherichia coli and Pseudomonas fluorescens recipients at frequencies similar to those observed with A. tumefaciens recipients. Rhizobium meliloti harboring the binary system also transferred the oriT/tra component to these recipients. However, E. coli or P. fluorescens donors harboring the binary system did not transfer pFRtra to any of the recipients. Furthermore, while the A. tumefaciens and R. meliloti donors produced high levels of the autoinducer, the P. fluorescens and E. coli donors produced only trace amounts of this signal molecule. These results indicate that the tra system of pTiC58 is fully contained within the characterized tra and trb regions of the Ti plasmid, that conjugation does not require functions encoded by the vir system for maximal activity, and that while the Ti plasmid tra system recognizes diverse gram-negative bacteria as recipients, of the hosts tested, it functions only in members of the family Rhizobiaceae.