This study was conducted to evaluate the potential applicability of an in situ biological reactive barrier system to treat nitrate-contaminated bank filtrate. The reactive barrier consisted of sulfur granules as an electron donor and autotrophic sulfur-oxidizing bacteria as a biological component. Limestone was also used to provide alkalinity. The results showed that the autotrophic sulfur oxidizers were successfully colonized on the surfaces of the sulfur particles and removed nitrate from synthetic bank filtrate. The sulfur-oxidizing activity continuously increased with time and then was maintained or slightly decreased after five days of column operation. Maximum nitrate removal efficiency and sulfur oxidation rate were observed at near neutral pH. Over 90% of the initial nitrate dissolved in synthetic bank filtrate was removed in all columns tested with some nitrite accumulation. However, nitrite accumulation was observed mainly during the initial operation period, and the concentration markedly diminished with time. The nitrite concentration in effluent was less than 2 mg-N/l after 12 days of column operation. When influent nitrate concentrations were 30, 40, and 60 mg-N/l and sulfur content in column was 75%, half-order autotrophic denitrification reaction rate constants were 31.73 x 10(-3), 33.3 x 10(-3), and 36.4 x 10(-3) mg(1/2)/l(1/2)min, respectively. Our data on the nitrate distribution profile along the column suggest that an appropriate wall thickness of a reactive barrier for autotrophic denitrification may be 30 cm when influent nitrate concentration is less than 60 mg-N/l.