Pathogenic Yersinia spp., including the etiological agent of plague, Y. pestis, all carry a common plasmid that encodes a number of essential virulence determinants, the Yop proteins. One of these, YopE, has been shown to be involved in the obstruction of the primary host defense by a molecular mechanism leading to inhibition of phagocytosis (R. Rosqvist, A. Forsberg, M. Rimpiläinen, T. Bergman, and H. Wolf-Watz, Mol. Microbiol. 4:657-667, 1990). Although the Yop proteins are secreted into the culture supernatant in vast amounts, in vitro studies of the function of the Yop proteins have so far been unsuccessful. We show that isolated Yop proteins indeed can cause cytotoxic effects in vitro if the proteins are introduced intracellularly into the eukaryotic cell. Isolated Yop proteins of Yersinia pseudotuberculosis were found to disrupt the microfilament structure when microinjected intracellularly into the host cell. In particular, YopE was demonstrated to be directly involved in the cytotoxic action, whereas YopD seems to have a critical role in translocating the YopE protein through the host cell membrane. These results elucidate the requirement for at least some of the Yop proteins to leave the pathogen during infection.