A single plasmid-encoded protein, the septal DNA translocator TraB, is sufficient to promote conjugal plasmid transfer in mycelial streptomycetes. To analyse the molecular mechanism of conjugation the closely related TraB proteins from plasmids pSG5 of Streptomyces ghanaensis and pSVH1 of Streptomyces venezuelae were characterized. TraB of pSG5 was expressed as a fusion protein with eGFP and found to be localized at the hyphal tips of Streptomyces lividans by fluorescence microscopy, which strongly indicates that conjugation takes place at the tips of the mating mycelium. The TraB protein of pSVH1 was heterologously expressed in S. lividans with an N-terminal strep-tagII and purified as a soluble protein to near homogeneity. The purified protein was shown to hydrolyse ATP and to bind to a 50 bp non-coding pSVH1 sequence containing a 14 bp direct repeat. The protein-DNA complex was too large to enter an agarose gel, indicating that multimers of TraB were bound to the DNA. Denaturation of the protein-DNA complex released unprocessed plasmid DNA demonstrating that the TraB protein does not possess nicking activity. Our experimental data provide evidence that conjugal DNA transfer in streptomycetes is mediated by the septal DNA translocator TraB, an plasmid-encoded ATPase that interacts non-covalently with DNA and translocates an unprocessed double-stranded DNA molecule at the hyphal tip into the recipient.