Pathogenic yersiniae (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) harbor a 70-kb virulence plasmid (pYV) that encodes a type III secretion system and a set of at least six effector proteins (YopH, YopO, YopP, YopE, YopM, and YopT) that are injected into the host cell cytoplasm. Yops (Yersinia outer proteins) disturb the dynamics of the cytoskeleton, inhibit phagocytosis by macrophages, and downregulate the production of proinflammatory cytokines, which makes it possible for yersiniae to multiply extracellularly in lymphoid tissue. Y. enterocolitica serotype O:8 belongs to the highly mouse-pathogenic group of yersiniae in contrast to Y. enterocolitica serotype O:9. However, there has been no systematic study of the contribution of Yops to the pathogenicity of Y. enterocolitica O:8 in mice. We generated a set of yop gene deletion mutants of Y. enterocolitica O:8 by using the novel Red cloning procedure. We subsequently analyzed the contribution of yopH, -O, -P, -E, -M, -T, and -Q deletions to pathogenicity after oral and intravenous infection of mice. Here we showed for the first time that a DeltayopT deletion mutant colonizes mouse tissues to a greater extent than the parental strain. The DeltayopO, DeltayopP, and DeltayopE mutants were only slightly attenuated after oral infection since they were still able to colonize the spleen and liver and cause systemic infection. The DeltayopO mutant was lethal for mice, whereas DeltayopP and DeltayopE mutants were successfully eliminated from the spleen and liver 2 weeks after infection. In contrast the DeltayopH, DeltayopM, and DeltayopQ mutants were highly attenuated and not able to colonize the spleen and liver on any of the days tested. The DeltayopH, DeltayopO, DeltayopP, DeltayopE, DeltayopM, and DeltayopQ mutants had only modest defects in the colonization of the small intestine and Peyer's patches. The DeltayopE mutant was eliminated from the small intestine 3 weeks after infection, whereas the DeltayopH, DeltayopP, DeltayopM, and DeltayopQ mutants continued to colonize the small intestine at this time.