Mononuclear phagocytes can be used by intracellular pathogens to disseminate throughout the host. In the bloodstream these cells are generically referred to as monocytes. However, blood monocytes are a heterogeneous population, and the exact identity of the leukocyte (s) relevant for microbial spreading is not known. Experiments reported in this study used Listeria monocytogenes-infected mice to establish the phenotype of parasitized blood leukocytes and to test their role in systemic dissemination of intracellular bacteria. More than 90% of the blood leukocytes that were associated with bacteria were CD11b+ mononuclear cells. Analysis of newly described monocyte subsets showed that most infected cells belonged to the Ly-6C high monocyte subset and that Ly-6C high and Ly-6C neg-low monocytes harbored similar numbers of bacteria per cell. Interestingly, systemic infection with wild-type or ΔactA mutants of L. monocytogenes, both of which escape from phagosomes and replicate intracellularly, caused expansion of the Ly-6C high subset. In contrast, this was not evident after infection with Δhly mutants, which neither escape phagosomes nor replicate intracellularly. Importantly, when CD11b+ leukocytes were isolated from the brains of lethally infected mice, 88% of these cells were identified as Ly-6C high monocytes. Kinetic analysis showed a significant influx of Ly-6C high monocytes into the brain 2 days after systemic infection. This coincided with both bacterial invasion and up-regulation of brain macrophage chemoattractant protein-1 gene expression. These data indicate that the Ly-6C high monocyte subset transports L. monocytogenes into the brain and establish their role as Trojan horses in vivo.