The effects of penicillin G on the number and activity of nitrite-oxidizing bacteria were investigated in laboratory-scale reactors and batch tests. At a concentration of 100 mg L-1, addition of penicillin G for short periods did not significantly affect nitrite oxidation, while addition for more than 2 months suppressed nitrite oxidation. Fluorescence in situ hybridization with 16S ribosomal RNA-targeted probes revealed a slight decrease in the abundance of Nitrospira, while Nitrobacter was not affected by addition of penicillin G for more than 39 days. The resistance of nitrite-oxidizing bacteria to penicillin appeared to be positively affected by intermittent aeration only when accompanied by denitrification; otherwise, the aeration mode (continuous or intermittent aeration) did not significantly affect the abundance of Nitrobacter and Nitrospira.