This work is an attempt to elucidate the quantitative significance of acetaminophen (APAP) nitration in nitrifying activated sludge and to propose a reaction mechanism for this process. The link between nitrification and nitration of APAP was investigated at different scales. Results from field studies showed the occurrence of 3-nitro-APAP and to a lesser extent 3-chloro-5-nitro-APAP at concentration levels in the 50-300 ng/L range in effluents of a full scale wastewater treatment plant (WWTP) operated with nitrogen removal, whereas 3-hydroxy-APAP was eliminated after the nitrification step. Batch experiments with nitrifying activated sludge confirmed APAP transformation by nitration and suggested that nitrifying bacteria may play a role in this transformation process through the release of reactive nitrogen species. In vitro assays provided evidence that nitration through the production of nitrous acid is a very unlikely pathway. In contrast, nitric oxide (*NO) produced by nitrifying bacteria is probably involved in APAP nitration through the formation of peroxynitrite in presence of superoxide anion. The production of 3-nitro-APAP would only account for a few percents of the total transformation rate of APAP in WWTPs. The production of nitrated derivatives is highly relevant because of the potential ecotoxicological risks of these compounds.