pUDKH, a new plasmid DNA comprising human hepatocyte growth factor (HGF), was developed as a potential genetherapeutic product for peripheral arterial disease. An anion-exchange high performance liquid chromatographic (HPLC) method was developed to determine the proportion of plasmid DNA in both opened and supercoiled forms. A TSK gel DNA-NPR column was equilibrated with 50% buffer A (20 mmol/L Tris-HCl, pH 8.8) and 50% buffer B (buffer A with 1 mol/L NaCl added). pUDKH was analyzed by using a gradient HPLC with a mobile phase from 50% B to 80% B and with a UV detection at 260 nm. The flow rate was 0.5 mL/min. The method was also used to monitor the production procedures.