The 70-kb virulence plasmid, pYV, of Yersinia enterocolitica encodes a number of secreted proteins (Yops) which are essential for virulence. YopD, the 33-kDa product of the lcrGVHyopBD operon, appears to be involved in delivering YopE and YopH (the Yersinia protein tyrosine phosphatase) into target cells. These proteins then act in concert to cause cytotoxicity in host cells. Previously, we reported that bacteria carrying transposon insertions in yopD are not cytotoxic for macrophages, show impaired tyrosine phosphatase activity in host cells, and are avirulent for mice (E. L. Hartland, S. P. Green, W. A. Phillips, and R. M. Robins-Browne, Infect. Immun. 62:4445-4453, 1994). trans complementation of yopD mutants of Y. enterocolitica with the yopD gene restores all these properties. In this study, we show that polar mutations in proximal genes of the lcrGVHyopBD operon also abrogated bacterial virulence and the capacity to induce cytotoxicity in mouse bone marrow-derived macrophages and HEp-2 epithelial cells. Moreover, trans complementation of a yopBD mutant with the yopD gene alone was not sufficient to restore the ability of the bacteria to cause cytotoxicity. Further work showed that YopB was required for cytotoxicity, dephosphorylation of host proteins, and virulence for mice. These findings indicate that YopB and YopD may serve a related function in Y. enterocolitica and that they may act together to deliver intracellularly acting Yops to their respective targets in host cells.