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The Agrobacterium tumefaciens Ti plasmid virulence (vir) region contains at least six transcriptional units required for the efficient transfer of T-DNA to the plant genome (virA, B, C, D, E, and G). We have reported that two proteins encoded by the 5'portion of the virD operon are required for a site-specific endonuclease activity that nicks the direct repeats which flank the T-DNA. We have presented the nucleotide sequence for this portion of the operon. The nucleotide sequence of the remainder of the virD operon essential for virulence has now been determined. Two additional open reading frames encode proteins of 21.3 and 75.8 kilodaltons (kd). Translational fusions between virD2, virD3, and virD4 proteins and trpE produced fusion proteins of the size predicted from the nucleotide sequence data. We have used antisera directed against the trpE-virD2 fusion protein to detect both native virD2 protein and a virD2-lacZ fusion protein in crude extracts from Agrobacterium.