YopH is translocated by cell-surface-bound bacteria through the plasma membrane to the cytosol of the HeLa cell. The transfer mechanism is contact dependent and polarizes the translocation to only occur at the contact zone between the bacterium and the target cell. More than 99% of the PTPase activity is associated with the HeLa cells. In contrast to the wild-type strain, the yopBD mutant cannot deliver YopH to the cytosol. Instead YopH is deposited in localized areas in the proximity of cell-associated bacteria. A yopN mutant secretes 40% of the total amount of YopH to the culture medium, suggesting a critical role of YopN in regulation of the polarized translocation. Evidence for a region in YopH important for its translocation through the plasma membrane of the target cell but not for secretion from the pathogen is provided.