Biological autotrophic sulfur oxidation processes have been proposed to remove heavy metals from wastewater treatment sludge by bioleaching. We made a characterization of the microbial population in batch and continuous sludge bioleaching reactors using fluorescent in situ hybridization of fluorescently-labeled oligonucleotidic probes targeting rRNA in a 'top to bottom approach'. Batch incubations of sludge with 0.2% (w/v) elemental sulfur resulted in a pH value of 5. Alpha-Proteobacteria hybridizing with probe ALF1b were dominant in this incubation. Members of the Acidophilium-group (hybridizing with probe Acdp821) of Nitrospira/Leptospirillum phylum (Ntspa712 probe) and from the archaeal domain (ARCH915) were also detected. When sludge was incubated with 1% elemental sulfur in batch or continuous reactor experiments, final pH values were always below 2. Active microbial communities consisted almost exclusively of gamma-Proteobacteria (hybridizing with probe GAM42a). However, further hybridization experiments with probe Thio820 targeting Acidithiobacillus ferroxidans and Acidithiobacillus thioxidans gave negative results. A new probe, named THIO181, encompassing all known members of the genus was designed. Hybridization perfomed with THIO181 and GAM42a showed a perfect co-localization of the hybridization signals. Further hybridization experiments with probe THIO181 and THC642, specific for the species Acidithiobacillus caldus, confirmed that this bacteria was largely responsible for the sulfur oxidation reaction in our acidophilic sludge bioleaching reactors.