Yersinia pseudotuberculosis initiates systemic disease after translocation across the intestinal epithelium. Three Y. pseudotuberculosis factors, previously identified by their ability to promote association with cultured cells, were evaluated for their relative roles in translocation. To this end, mutants defective for invasin, YadA, or pH 6 antigen were tested for movement from the intestinal lumen into the subepithelium. Within 45 min after introduction of bacteria into the lumen, wild-type bacteria were found in the Peyer's patch. Mutants expressing defective invasin derivatives were unable to promote efficient translocation into the Peyer's patch and instead colonized on the luminal surface of the intestinal epithelium. In particular, a translocation defect was observed in a Y. pseudotuberculosis strain that expressed an uptake-defective invasin protein retaining considerable receptor binding activity. To attempt to reduce binding to luminal mucus, Y. pseudotuberculosis yadA and inv yadA strains were analyzed. Both strains had reduced mucus binding, with the inv yadA mutant revealing an alternate uptake pathway that was invasin independent. A mutant defective in the production of the pH 6 antigen adhesin also showed reduced binding to luminal mucus, with specific localization of bacteria in M cells. These results indicate that Y. pseudotuberculosis adhesive factors control the site of bacterial interaction within the intestinal environment and that loss of one factor causes drastic changes in the preferred site of localization of the bacterium in this locale.