Anaerobic batch and flow-through experiments were performed to confirm the role of pyrite as electron donor in bacterial denitrification and to look into the feasibility of pyrite-driven denitrification of nitrate-contaminated groundwater. Nitrate reduction was satisfactorily accomplished in experiments with pyrite as the sole electron donor, in presence of the autotrophic denitrifying bacterium Thiobacillus denitrificans and at nitrate concentrations comparable to those observed in contaminated groundwater. The experimental results corroborated field studies in which the reaction occurred in aquifers. Nitrate reduction rates and nitrate removal efficiencies were dependent on pyrite grain size, initial nitrate concentration, nitrate-loading rate and pH. The N and O isotopic enrichment factors (epsilon N and epsilon O) obtained experimentally for pyrite-driven nitrate reduction by T. denitrificans ranged from -13.5 parts per thousand to -15.0 parts per thousand and from -19.0 parts per thousand to -22.9 parts per thousand, respectively. These values indicated the magnitude of the isotope fractionation that occurs in nitrate-contaminated aquifers dominated by autotrophic denitrification. (C) 2010 Elsevier B.V. All rights reserved.