DNA regulatory motifs reflect the direct transcriptional interactions between regulators and their target genes and contain important information regarding transcriptional networks. In silico motif detection strategies search for DNA patterns that are present more frequently in a set of related sequences than in a set of unrelated sequences. Related sequences could be genes that are coexpressed and are therefore expected to share similar conserved regulatory motifs. We identified coexpressed genes by carrying out microarray-based transcript profiling of Salmonella enterica serovar Typhimurium in response to the spent culture supernatant of the probiotic strain Lactobacillus rhamnosus GG. Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. They are known to antagonize intestinal pathogens in vivo, including salmonellae. S. enterica serovar Typhimurium causes human gastroenteritis. Infection is initiated by entry of salmonellae into intestinal epithelial cells. The expression of invasion genes is tightly regulated by environmental conditions, as well as by many bacterial factors including the key regulator HilA. One mechanism by which probiotics may antagonize intestinal pathogens is by influencing invasion gene expression. Our microarray experiment yielded a cluster of coexpressed Salmonella genes that are predicted to be down-regulated by spent culture supernatant. This cluster was enriched for genes known to be HilA dependent. In silico motif detection revealed a motif that overlaps the previously described HilA box in the promoter region of three of these genes, spi4_H, sicA, and hilA. Site-directed mutagenesis, beta-galactosidase reporter assays, and gel mobility shift experiments indicated that sicA expression requires HilA and that hilA is negatively autoregulated.