Partial nitrification of ammonium to nitrite under oxic conditions (nitritation) is a critical process for the effective use of alternative nitrogen removal technologies from wastewater. Here we investigated the conditions which promote establishment of a suitable microbial community for performing nitritation when starting from regular sewage sludge. Reactors were operated in duplicate under different conditions (pH, temperature, and dilution rate) and were fed with 50 mM ammonium either as synthetic medium or as sludge digester supernatant. In all cases, stable nitritation could be achieved within 10 to 20 days after inoculation. Quantitative in situ hybridization analysis with group-specific fluorescent rRNA-targeted oligonucleotides (FISH) in the different reactors showed that nitrite-oxidizing bacteria of the genus Nitrospira were only active directly after inoculation with sewage sludge (up to 4 days and detectable up to 10 days). As demonstrated by quantitative FISH and restriction fragment length polymorphism (RFLP) analyses of the amoA gene (encoding the active-site subunit of the ammonium monooxygenase), the community of ammonia-oxidizing bacteria changed within the first 15 to 20 days from a more diverse set of populations consisting of members of the Nitrosomonas communis and Nitrosomonas oligotropha sublineages and the Nitrosomonas europaea-Nitrosomonas eutropha subgroup in the inoculated sludge to a smaller subset in the reactors. Reactors operated at 30 degrees C and pH 7.5 contained reproducibly homogeneous communities dominated by one amoA RFLP type from the N. europaea-N. eutropha group. Duplicate reactors at pH 7.0 developed into diverse communities and showed transient population changes even within the ammonia oxidizer community. Reactors at pH 7.5 and 25 degrees C formed communities that were indistinguishable by the applied FISH probes but differing in amoA RFLP types. Communities in reactors fed with sludge digester supernatant exhibited a higher diversity and were constantly reinoculated with ammonium oxidizers from the supernatant. Therefore, such systems could be maintained at a higher dilution rate (0.75 day(-1) compared to 0.2 day(-1) for the synthetic wastewater reactors). Despite similar reactor performance with respect to chemical parameters, the underlying community structures were different, which may have an influence on stability during perturbations.