During infection of its hosts, Salmonella enters intestinal epithelial cells. Many Salmonella typhimurium genes required for bacterial entry into host cells are encoded on a 40 kb 'pathogenicity island'. We report here the identification of hilA, a gene within the 'island' that appears to encode an activator of invasion gene expression. By using a set of lacZY transcriptional fusions to S. typhimurium invasion genes, we found that hilA activates the expression of invasion genes located on the 'pathogenicity island'. hilA is required for efficient entry into HEp-2 cells in vitro. The predicted amino acid sequence of hilA shares significant homology with the DNA-binding domains of the OmpR-ToxR family of transcriptional activators. However, unlike OmpR and ToxR, HilA contains neither a phosphoryl acceptor nor a membrane-spanning domain, and, therefore, its activity may be modulated by a novel mechanism. Many environmental conditions modulate the ability of Salmonella to enter non-phagocytic mammalian cells. It has been proposed that induction of Salmonella invasion proteins in response to a combination of environmental cues ensures that bacterial entry is limited to specific sites and times during infection. Our results are consistent with the hypothesis that hilA plays a key role in the regulation of Salmonella invasion during infection.