Three sequencing batch reactors (SBRs) labeled with R(Ahead), R(Exact) and R(Exceed) were employed to investigate the synergetic inhibition effect of free ammonia (FA) and length of aeration phase on the activity of ammonia-oxidizing bacteria ( AOB) and nitrite- oxidizing bacteria (NOB) after shortcut nitritation was achieved in the systems. The experiments were conducted under the conditions of three FA concentrations (0.5, 5. 1, 10.1 mg · L⁻¹) combined with three kinds of aeration time (t(Exact): the time when ammonia oxidation was completed; t(Ahead): 30 min ahead of the time when ammonia oxidation was completed; t(Exceed): 30 min exceeded when the time ammonia oxidation was completed). It was found that short-cut nitrification could be successfully established in three reactors with a FA level of 10.1 mg · L⁻¹. Meanwhile, the speed of achieving nitritation was in the sequence of R(Ahead) > R(Exact) > R(Exceed) with operational cycles of 56, 62 and 72, respectively. Compared to AOB, NOB in the three reactors was observed to be more sensitive to FA, resulting in AOB activity higher than NOB activity throughout the whole experimental period. Moreover, there was great difference in the activity coefficient ( η) between AOB and NOB. The activity coefficients of AOB were in the order of η(RExact) > η(RExceed) > η(RAhead) with the values of 104.4%, 100% and 85.8%, respectively. Nevertheless, the activity coefficients of NOB were in the order of η(RExceed) > η(RExact) > η(RAhead) with the values of 71.2%, 64.9% and 50.2%, respectively.