We have identified several environmental isolates of Salmonella senftenberg and S. litchfield which carry a deletion encompassing a vast segment of the centisome 63 region of the Salmonella chromosome. The deletion includes the entire inv, spa, and hil loci, which are required for entry of Salmonella spp. into mammalian cells. Consequently, these isolates were found to be markedly deficient in the ability to enter cultured epithelial cells. In contrast, no deletions were found in the corresponding regions of the chromosomes of clinical isolates of these serovars; consequently, these isolates were found to be highly invasive for cultured epithelial cells. These data confirm the importance of the centisome 63 region of the Salmonella chromosome in mediating the entry of these organisms into cultured mammalian cells and indicate that additional entry pathways are presumably not utilized by these environmental isolates. These results are also consistent with the notion that this region constitutes a pathogenicity island which remains unstable in certain Salmonella serotypes.