In the Bacillus subtilis phosphorelay signal transduction system for sporulation initiation, signals competing with the differentiation process are interpreted by aspartyl-phosphate phosphatases that specifically dephosphorylate the Spo0F or Spo0A response regulators. The RapA phosphatase is regulated by the PhrA pentapeptide that directly and specifically inhibits its activity. PhrA specificity for RapA inhibition is dependent upon the amino acid sequence of the peptide. Here we show that the pentapeptide affinity for the phosphatase requires a free carboxylate group at the C-terminal amino acid. A free C-terminal carboxylic acid PhrA pentapeptide inhibits RapA phosphatase activity at a 1:1 ratio and it is approximately 200 fold more active than a C-terminal amide peptide. Therefore, coordination of the terminal carboxylate group appears to be critical for peptide binding to RapA.