Nitrate contamination is becoming a widespread environmental problem, and autotrophic denitrification with Thiobacillus denitrificans is a promising process considering efficiency, cost and maintenance. The denitrification efficiencies of T. denitrificans were compared in batch reactors between free cells and cells immobilized on polyvinyl alcohol (PVA) carriers made with thrice freezing/thawing and boric acid methods. The results indicated that the free cell reactor of T. denitrificans added with 10% (v/v) of PVA carrier made by thrice freezing/thawing (PVA-TFT) exhibited faster in S(2)O(3)(2-)-S consumption, SO(4)(2-) generation, and NO(3)(-)-N denitrification, with corresponding values being 165 mg (S(2)O(3)(2-)-S)/L.d, 491 mg (SO(4)(2-))/Ld, and 44 mg (NO(3)(-)-N)/Ld, which were increased by 50%, 61%, and 57% respectively compared to the control reactor with only free cells. Inhibition of denitrification by accumulated SO(4)(2-) in PVA-TFT reactor appeared at the concentration of approximately 6000 mg (SO(4)(2-))/L, and 75% of NO(3)(-)-N removal efficiency was achieved after 12d operation under the condition of initial 700 mg/L NO(3)(-)-N concentration.