Shigella flexneri invades eucaryotic cells and grows in the cytoplasm. Lysis of the phagosomal membrane is a prerequisite for both intracellular multiplication and movement of the bacteria that gain direct access to the host cell actin. In HeLa cells, bacteria generate their own movement essentially by inducing actin polymerization. Polymerization of actin enables them to move rapidly and randomly in the cytoplasm and to spread from one cell to another through protrusions of the host cell membrane. This movement was designated the Ics phenotype. In contrast, in chicken embryo fibroblasts, bacteria move along actin filaments in a very organized manner, following the cytoskeletal architecture; this movement was designated the Olm phenotype. Bacterial movement is a major virulence factor in that it is necessary for efficient colonization of the intestinal epithelium of infected macaque monkeys. Further characterization of the cellular events that lead to colonization of the colonic intestinal epithelium was needed. In order to characterize the movement in vitro in a cell assay system more closely related to the intestinal epithelium, we used human colonic epithelial Caco-2 cells. The movement of bacteria as observed by using immunofluorescence and confocal microscopy appeared to result from the expression of both the Olm and Ics phenotypes. The former allowed colonization of cells along the actin filament ring of the perijunctional area. The latter promoted passage from one cell to adjacent cells. This in vitro pattern of movement and multiplication gives S. flexneri, once it has entered an epithelial cell, the unique capacity to spread through the entire epithelial layer without having further contact with the extracellular compartment.