Bacterial conjugation is a DNA transfer event that requires three plasmid-encoded multi-protein complexes: the membrane-spanning mating pair formation (Mpf) complex, the cytoplasmic nucleoprotein relaxosome complex, and a homo-multimeric coupling protein that links the Mpf and relaxosome at the cytoplasmic membrane. Bacterial two-hybrid (BTH) technology and immunoprecipitation were used to demonstrate an interaction between the IncH plasmid-encoded transfer protein TraJ and the coupling protein TraG. TraJ is essential for conjugative transfer but is not required for the formation of the conjugative pilus, and is therefore not regarded as an Mpf component. Fractionation studies indicated that TraJ shared a similar cellular domain to that of TraG at the cellular membrane. Protein BLAST analyses have previously identified TraJ homologues encoded in a multitude of plasmid and chromosomal genomes that were also found to encode an adjacent TraG homologue, thus indicating co-inheritance. BTH analysis of these TraJ and cognate TraG homologues demonstrated conservation of the TraJ-TraG interaction. Additional occurrences of the traJ-traG module were also detected in genomic sequence data throughout the Proteobacteria, and phylogenetic comparison of these IncH-like TraG proteins with the coupling proteins encoded by other conjugative transfer systems (including IncP, IncW and IncF) that lack TraJ homologues indicated that the H-like coupling proteins were distinct. Accordingly, the IncP, IncW and IncF coupling proteins were unable to interact with TraJ, but were able to interact with IncH plasmid-encoded TrhB, an Mpf component known to complex with its cognate coupling protein TraG. The divergence of the IncH-type coupling proteins may partly be due to the requirement of TraJ interaction, and notably, TraG and TraJ cumulatively represent the domain architecture of the known translocase family FtsK/SpoIIIE. It is proposed that TraJ is a functional part of the IncH-type coupling protein complex required for translocation of DNA through the cytoplasmic membrane.