Invasion of the intestinal epithelium is thought to be an important step in the pathogenesis of Salmonella infections. Using an in vitro system, we have isolated a genetic locus, inv, that confers to a noninvasive strain of Salmonella typhimurium the ability to penetrate tissue culture cells. Highly virulent S. typhimurium strains carrying inv mutations were defective for entry into Henle-407 cells while remaining unaffected in their ability to attach to cultured cells. When administered perorally to BALB/c mice, inv mutants of S. typhimurium had higher 50% lethal doses (LD50) than their wild-type parent strains. To the contrary, there were no differences in the observed LD50 when strains were administered intraperitoneally. In addition, inv mutants presented decreased ability to colonize the Peyer's patches, the small intestinal wall, and the spleen when administered perorally, although when administered intraperitoneally, they showed no difference in their ability to colonize the spleen compared to the wild-type parent strain.