The Des pathway of Bacillus subtilis regulates the synthesis of the cold-shock induced membrane-bound enzyme Delta5-fatty acid desaturase (Delta5-Des). A central component of the Des pathway is the response regulator, DesR, which is activated by a membrane-associated kinase, DesK, in response to a decrease in membrane lipid fluidity. Despite genetic and biochemical studies, specific details of the interaction between DesR and the DNA remain unknown. In this study we show that only the phosphorylated form of protein DesR is able to bind to a regulatory region immediately upstream of the promoter of the Delta5-Des gene (Pdes). Phosphorylation of the regulatory domain of dimeric DesR promotes, in a cooperative fashion, the hierarchical occupation of two adjacent, non-identical, DesR-P DNA binding sites, so that there is a shift in the equilibrium toward the tetrameric active form of the response regulator. Subsequently, this phosphorylation signal propagation leads to the activation of the des gene through recruitment of RNA polymerase to Pdes. This is the first dissected example of a transcription factor functioning as a phosphorylation-activated switch for a cold-shock gene, allowing the cell to optimize the fluidity of membrane phospholipids.