Virulent Yersinia species harbor a common plasmid that encodes essential virulence determinants (Yersinia outer proteins [Yops]), which are regulated by the extracellular stimuli Ca2+ and temperature. The V-antigen-encoding operon has been shown to be involved in the Ca(2+)-regulated negative pathway. The genetic organization of the V-antigen operon and the sequence of the lcrGVH genes were recently presented. The V-antigen operon was shown to be a polycistronic operon having the gene order lcrGVH-yopBD (T. Bergman, S. Håkansson, A. Forsberg, L. Norlander, A. Macellaro, A. Bäckman, I. Bölin, and H. Wolf-Watz, J. Bacteriol. 173:1607-1616, 1991; S. B. Price, K. Y. Leung, S. S. Barve, and S. C. Straley, J. Bacteriol. 171:5646-5653, 1989). We present here the sequence of the distal part of the V-antigen operons of Yersinia pseudotuberculosis and Yersinia enterocolitica. The sequence information encompasses the yopB and yopD genes and a downstream region in both species. We conclude that the V-antigen operon ends with the yopD gene. This conclusion is strengthened by the observation of an insertion-like element downstream of the yopD gene. The translational start codons of YopB and YopD have been identified by N-terminal amino acid sequencing. By computer analysis, the yopB and yopD gene products were found to be possible transmembrane proteins, and YopD was shown to contain an amphipathic alpha-helix in its carboxy terminus. These findings contrast with the general globular pattern observed for other Yops. Homology between Yersinia LcrH and Shigella flexneri IppI and between Yersinia YopB and S. flexneri IpaB was found, suggesting conservation of this locus between these two genera. YopB was also found to have a moderate level of homology, especially within the hydrophobic regions, to members of the RTX protein family of alpha-hemolysins and leukotoxins, indicating that YopB might exhibit a similar function.