The process of bacterial conjugation involves the unidirectional transfer of a conjugative plasmid between bacteria. To initiate this process, a single strand of the plasmid is cleaved by the plasmid-encoded endonuclease, the relaxase. The relaxase for the F plasmid system is Tral, a 192 kDa bifunctional protein known to specifically bind and cleave ssDNA. It was previously shown that the protein bound to its cognate ssDNA recognition sequence with sub-nanomolar affinity and specificity. However, the molecular details of this interaction were not well understood. In order to better understand the nature of the ssDNA recognition, binding and cleavage, we initiated structural and biochemical studies of the N-terminal relaxase domain of the protein, TraI36. The X-ray crystallographic structure revealed that the protein employs a unique ssDNA recognition strategy involving extensive shape complementarity and DNA-DNA interactions to define specificity. Mutagenesis results supported the importance of residues within a flap region that folded over the bound ssDNA and also of the shape complementarity between protein and ssDNA. Studies of the active site residues by characterization of the ssDNA cleavage activity and metal binding affinity of the mutants has begun to elucidate the roles these residues in bacterial conjugation.