Kinetics of sulphur-limestone autotrophic denitrification process in batch assays and the impact of sulphur/limestone ratio on the process performance in long-term operated packed-bed bioreactors were evaluated. The specific nitrate and nitrite reduction rates increased almost linearly with the increasing initial nitrate and nitrite concentrations, respectively. The process performance was evaluated in three parallel packed-bed bioreactors filled with different sulphur/limestone ratios (1:1, 2:1 and 3:1, v/v). Performances of the bioreactors were studied under varying nitrate loadings (0.05 - 0.80 gNO(-)(3) - NL⁻¹ d⁻¹) and hydraulic retention times (3-12 h). The maximum nitrate reduction rate of 0.66 g L⁻¹ d⁻¹ was observed at the loading rate of 0.80 g NO(-)(3) - N L⁻¹ d⁻¹ in the reactor with sulphur/limestone ratio of 3:1. Throughout the study, nitrite concentrations remained quite low (i.e. below 0.5 mg L⁻¹ NO(-)(2) -N. The reactor performance increased in the order of sulphur/limestone ratio of 3:1, 2:1 and 1:1. Denaturing gradient gel electrophoresis analysis of 16S rRNA genes showed quite stable communities in the reactors with the presence of Methylo virgulaligni, Sulfurimonas autotrophica, Sulfurovum lithotrophicum, Thiobacillus aquaesulis and Sulfurimonas autotrophica related species.