The cynR protein, which is a positive regulator for cynTSX operon in the presence of cyanate as well as a negative regulator for its own gene, was overexpressed by the T7 RNA polymerase/T7 promoter expression system in which the cynR gene was cloned under the control of T7 promoter. The cynR protein was purified on the serial columns of DEAE-cellulose, phosphocellulose, and Sephadex G-200. Throughout the purification, the cynR protein was identified as a single band of 32,000 daltons on SDS-polyacrylamide gel. The molecular weight of the protein in the native state was determined to be 64,000 daltons by HPLC, suggesting that the cynR protein exists as a dimer. The N-terminal acid sequence of the purified cynR protein was essentially the same as those deduced from the DNA sequence of cynR gene. In the gel retardation assay, the regulatory protein was specifically bound to the intergenic region between the cynTSX operon and the cynR gene. This result suggests that cynR protein binds to the intergenic region and regulates the expression of cynTSX structural genes and cynR regulatory gene simultaneously.