Three identical lab-scale biofilters were employed to optimize the start-up process for simultaneous removal of iron (Fe), manganese (Mn), and ammonia from potable water supplies. Nitrifying sludge and backwashing sludge containing Mn-oxidizing bacteria (MnOB) were used as the inocula. The start-up strategies consisted of simultaneous and separate inoculation of the two kinds of sludge. The influent Fe was removed immediately when the biofilters began to operate. The effects of nitrification for ammonia removal showed no significant difference between these biofilters. However, the beginning of Mn removal with separate inoculation was faster than that of simultaneous inoculation. The Mn removal can be described by using first order reaction; and the k (rate constant, min(1)) values were 0.147 +/- 0.007 (mean +/- standard deviation) and 0.153 +/- 0.006. Besides the commonly reported MnOB genus Crenothrix, MnOB genera were also found to be related to the genera rarely reported in the potable water treatment systems. (C) 2014 Elsevier Ltd. All rights reserved.