Synthesis of alginate by Pseudomonas aeruginosa correlates with its pathogenicity in the lungs of patients suffering from cystic fibrosis (CF). Alginate synthesis-encoding genes (alg) in P. aeruginosa are normally silent, but are specifically triggered in the CF lung environment. The promoter for the algD gene, located at the upstream end of the alg cluster, is activated by environmental factors such as high osmolarity, nutrient limitation and dehydration. Several regulatory proteins are known to control transcription from the algD promoter. Among these proteins is AlgR1 which is homologous to the phosphorylation-dependent response regulators of the two-component signal transduction system. In this paper, we report that AlgR2, an 18-kDa protein which in cooperation with AlgR1 regulates the algD promoter, undergoes phosphorylation in the presence of ATP. The phosphate group acquired by AlgR2 is then transferred to AlgR1. In addition, we show that AlgR1 can be phosphorylated by an AlgR2-analog in Escherichia coli. AlgR1 is isolated in a phosphorylatable 80-kDa complex in association with AlgR2 in P. aeruginosa and the AlgR2-analog in E. coli.