This study investigated the chronic impact of sulfamethoxazole (SMX) on activated sludge sustaining an enriched nitrifying biomass. For this purpose, a laboratory scale fill and draw reactor was operated with 100 mg COD/L of peptone mixture and 50 mg N/L of ammonia at a sludge age of 15 days. Additionally, the biomass was exposed to a daily SMX dose of 50 mg N/L once the reactor reached steady-state conditions. The reactor performance and microbial composition were monitored for 37 days with conventional parameters and molecular techniques based on the gene for ammonia monooxygenase subunit A (amoA) and the prokaryotic 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene cloning analyses suggested a microbial community change concurrent with the addition of SMX. Specifically, quantitative polymerase chain reaction analyses (qPCR/RT-qPCR) revealed a significant reduction in the levels and activity of ammonia oxidizing bacteria (AOB). However, the acclimation period ended with high amoA mRNA levels and improved nitrification efficiency. Partial degradation of SMX by heterotrophic bacteria was also observed. (c) 2016 Elsevier Ltd. All rights reserved.