According to their host range, Salmonella enterica can be divided into generalists, host-adapted, and host-specific serovars. The generalist Salmonella enterica serovar Typhimurium causes disease in many animal species but host specific serovars cause disease only in one host. In this study, the hypothesis that macrophages contribute to Salmonella host specificity in vivo was tested in mice. The survival of Typhimurium and the host specific Salmonella enterica serovar Typhi were compared in mouse macrophage cell lines (RAW 264.7 and J774A.1), and murine peritoneal, spleen and bone marrow derived macrophages in vitro. Differences in survival observed for the two serovars in these in vitro studies could not distinguish host specific from generalist serovars. When BALB/c mice were infected in vivo i.p., equivalent levels of Typhimurium could be found in the peritoneum 0.5 and 4 hr post-infection but Typhi decreased drastically between these two time points. Both Typhi and Typhimurium induced neutrophil influx into the peritoneum and macrophages disappeared with both serovars. With either serovar, macrophages were shown to be the major cell type containing internalized bacteria after both 0.5 and 4 hr of infection. However, significantly lower numbers of viable Typhi were recovered from macrophages infected in vivo than Typhimurium. These data showed that macrophages were able to distinguish Typhi from Typhimurium when infected in vivo, suggesting that the in vivo environment is important for macrophages to play an important role in Salmonella host specificity