In Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, a secreted fatty acid signaling molecule known as diffusible signal factor (DSF) is required for virulence and growth on low-iron medium. To identify other virulence-associated traits that are regulated by DSF in this pathogen, we have performed microarray analysis of transcriptional changes between the wild type and DSF-deficient mutants of X. oryzae pv. oryzae. Expression of genes that encode secreted hydrolytic enzymes, motility, and chemotaxis functions are negatively regulated by DSF while functions involved in adhesion and biofilm formation are positively regulated. Enzymatic assays for hydrolytic enzymes as well as assays for chemotaxis, motility, attachment, and biofilm formation corroborate these findings. These results demonstrate that, in X. oryzae pv. oryzae, DSF-mediated cell-to-cell signaling coordinates transition from solitary to biofilm lifestyle by promoting expression of attachment functions and negatively regulating expression of motility functions. This is in contrast to X. campestris pv. campestris, a pathogen of crucifers, wherein the DSF system positively regulates motility functions and negatively regulates biofilm formation. These results indicate that virulence-associated functions can be regulated in a completely contrasting fashion by the same signaling system in very closely related bacteria.