Ammonia-oxidizing bacteria (AOB) and anaerobic ammonia-oxidizing bacteria (AnAOB) are autotrophic microorganisms. Inorganic carbon (IC) is their main carbon source. The effects of IC limitation on AOB and AnAOB in the completely autotrophic nitrogen removal over nitrite (CANON) process in a sequencing batch biofilm reactor (SBBR) were examined. The optimal IC concentration in the influent was investigated. The start-up time of the CANON process from the activated sludge in the SBBR was 80 d under controlled free ammonia (FA) conditions and sufficient IC source. The AOB and AnAOB activities were limited by an IC concentration of 50 mg-C-L(-1) in the influent, whilst the nitrogen loading rate (NLR) was 200 mg-N x L(-1) x d(-1). The experiment on recovering the influent IC showed that AOB and AnAOB activities were affected by the IC limitation, and not by the pH or FA, at 200mg-N x L(-1) x d(-1) NLR and 50mg-C x L(-1) IC in the CANON process. The activities were recovered by increasing the IC concentration in the influent. From an economic point of view, the optimal IC concentration in the influent was 250mg-C x L(-1) at 200mg-N x L(-1) x d(-1) NLR in this CANON system.