Multiple yop mutant strains of Yersinia pseudotuberculosis not expressing several virulence effector Yop proteins (YopH, M, E, K and YpkA) were engineered. When high-copy-number plasmids carrying the ypkA or the yopE gene with their endogenous promoters were introduced into the engineered strains, the corresponding Yop protein was secreted at high levels in vitro. These multiple yop mutant strains, when harbouring the yopE gene in trans, behaved as the wild-type strain with respect to YopB-dependent translocation of YopE through the HeLa cell plasma membrane. Using these multiple yop mutant strains, it was demonstrated that the YpkA Ser/Thr protein kinase mediates morphological alterations of infected cultured HeLa cells different from those mediated by YopE and YopH. Furthermore, YpkA is shown to be translocated by a YopB-dependent translocation mechanism from surface-located bacteria and subsequently targeted to the inner surface of the target-cell plasma membrane. The pattern of YpkA localization after infection suggests that this Yop effector is involved in interference with signal transduction.