The hairy-root-inducing plasmid A4 (pRiA4) is capable of conferring tumorigenic symptoms on plants upon infection by its host bacterium, Agrobacterium rhizogenes. The virB operon on pRiA4 has been sequenced and found to be composed of 11 genes, virB1 to virB11, whose products mostly appear to be associated with the cell membrane. A novel structural characteristic is frequent overlappings between the translation termination and initiation codons of adjacent genes. This is indicative of fine tuning of relative translation frequencies for each VirB protein. These results support the view that VirB multisubunit complexes provide facilities for T-DNA transfer at the bacterial cell membrane. The structural organization of the pRiA4 virB operon was essentially identical to that of the previously reported virB operons of tumor-inducing plasmids, pTiC58 and pTiA6, and the corresponding VirB proteins of the three plasmids were extremely homologous to one another. On the basis of the structural similarity of each VirB protein, the phylogenetic relationship among pRiA4, pTiC58, and pTiA6 is discussed.