The traY gene product of plasmid R100 was purified as a hybrid protein, TraY-collagen-beta-galactosidase. The hybrid protein as well as the TraY' protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT. The oriT region consists of highly conserved and nonconserved regions among R100-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region. This supports the idea that the TraY protein has a role as a component of endonuclease in recognizing its own oriT sequence. Unexpectedly, however, the hybrid protein and the TraY' protein were also found to bind to two different AT-rich sequences (each 24 base pairs in length) in the promoter region preceding the traY gene (here called sbyB and sbyC). This suggests that the TraY protein may have another role in regulating the expression of its own gene. The "TAA(A/T)T" sequence motif observed in these binding sites might constitute a core sequence recognized by the TraY protein. Mg2+ is not required for the specific binding of the TraY protein.