A strain of Thiobacillus denitrificans was isolated after enrichment under anaerobic conditions by the continuous culture technique using thiosulfate as energy source and nitrate as electron acceptor and nitrogen source. The isolate was an active denitrifyer, the optimal conditions being 30°C and pH 7.5–8.0. Denitrification was inhibited by sulfate (the reaction product) above 5 g SO4=/l, whereas high concentrations of the substrates nitrate and thiosulfate were less harmful; nitrite affected denitrification above 0.2 g NO2−/l. During the time course of denitrification in a batch culture growth and substrate consumption slowed down already after only half the substrate was utilized due to product inhibition. The following parameters were determined in continuous culture under nitrate limitation: μmax=0.11 h−1, KS=0.2 mg NO3−/l, maximum denitrification rate=0.78 g NO3−/g cells·h, \(Y_{{\text{NO}}_{\text{3}} } = {\text{0}}{\text{.129}}\)g cells/g NO3−, \(Y_{{\text{S}}_{\text{2}} {\text{O}}_{\text{3}} } = {\text{0}}{\text{.085}}\)g cells/g S2O3=. Nitrite did not accumulate during steady state denitrification; the denitrification gas was almost pure N2. The concentrations of N2O and NO were below 1 ppm.