The abilities of Yersinia pestis to undergo restriction in Ca2+-deficient medium with concomitant production of V and W antigens (Vwa+) and to absorb exogenous pigments (Pgm+) are established virulence factors. Mutation of Y. pestis to Pgm- is known to promote resistance to pesticin (Pstr) and reduced lethality by peripheral routes of injection. Vwa+ Pgm- isolates of Y. pestis were shown in this study to retain virulence in mice when injected intravenously. Although Pgm- in appearance, wild-type cells of Yersinia pseudotuberculosis and Yersinia enterocolitica may also be sensitive to pesticin. Pstr mutants of Vwa+ strains of these species were similarly of reduced virulence, especially by peripheral routes of injection. The consequences of mutation to Vwa- and Pgm- or Pstr on growth and persistence in vivo were determined. After intravenous injection, Vwa+ yersiniae of all species exhibited sustained growth in mouse spleen, liver, and lung and accumulated in blood. Septicemia was not observed after similar injection of Vwa- mutants which were unable to maintain comparable rates of net increase in tissues. Mutation to Pgm- or Pstr did not influence proliferation but resulted in enhanced clearance from organs. It is known that reticuloendothelial cells serve as favored sites of replication for all wild-type yersiniae. Our results are consistent with the hypothesis that the Vwa+ phenotype favors growth within macrophages and that the Pgm+ and pesticin-sensitive phenotypes permit long-term, probably extracellular, retention within organs. Virulence in standard animal models (mice, rats, and guinea pigs) was not correlated with resistance to the bactericidal action of serum.