To characterize protein-DNA interactions involved in the initiation of conjugative transfer replication we isolated and dissected the transfer origins (oriT) of the promiscuous IncP plasmids RP4 and R751. Essential features of oriT are conserved: symmetric sequence repeats, the nic site and a pair of potential promoter sites that allow for divergent transcription of two tra operons. The relaxation nick and the end of a 19 bp inverted repeat are interspaced by eight basepairs. The 5'-terminal nucleotide at the nick is modified by an alkali-resistant residue and the 3'-nucleotide is accessible to extension by DNA polymerase I. Transfer gene products essential for the formation of the initiation complex (relaxosome) of conjugative DNA synthesis map adjacent to oriT. Two of these products, TraJ and TraK confer specificity to their homologous oriT exclusively. Proteins TraJ and TraK are the only components of the RP4 and R751 transfer machinery which cannot be interchanged. TraJ and at least two additional plasmid-encoded products are necessary for specific relaxation. The purified TraJ protein of RP4 possesses oriT-binding ability. The recognition sequence contains a palindromic sequence located within the right arm of the 19 bp inverted repeat. The TraJ binding site and the nic site are located on one side of the DNA double helix. We presume that this nucleoprotein structure is the initial complex in the pathway to the assembly of functional relaxosomes.