AbstractBiological reduction of nitric oxide (NO) to di‐nitrogen (N2) gas in aqueous Fe(II)EDTA2− solutions is a key reaction in BioDeNOx, a novel process for NOx removal from flue gases. The mechanism and kinetics of the first step of NO reduction, that is, the conversion of NO to N2O, was determined in batch experiments using various types of inocula. Experiments were performed in Fe(II)EDTA2− medium (5–25 mM) under BioDeNOx reactor conditions (55°C, pH 7.2 ± 0.2) with ethanol as external electron donor. BioDeNOx reactor mixed liquor gave the highest NO reduction rates (±0.34 ${\rm nmol}\,{\rm s}^{ - 1} \,{\rm mg}_{{\rm prot}}^{ - 1} $) with an estimated Km value for NO lower than 10 nM. The specific NO (to N2O) reduction rate depended on the NO (aq) and Fe(II)EDTA2− concentration as well as the temperature. The experimental results, complemented with kinetic and thermodynamic considerations, show that Fe(II)EDTA2−, and not ethanol, is the primary electron donor for NO reduction, that is, the BioDeNOx reactor medium (the redox system Fe(II)EDTA2−/Fe(III)EDTA−) interferes with the NO reduction electron transfer chain and thus enhances the NO denitrification rate. Biotechnol. Bioeng. 2008;100: 1099–1107. © 2008 Wiley Periodicals, Inc.