Activated sludge samples from municipal sewage treatment plants were characterized using 16S and 23S ribosomal RNA targeted oligonucleotide probes specific for defined phylogenetic groups of bacteria. Comparison of in situ community structures as determined by molecular biological methods with the composition of the heterotrophic saprophyte flora isolated on nutrient rich medium revealed large discrepancies. These are caused by the selectivity of media and culture conditions. The most significant effect of cultivation on nutrient rich medium is an underestimation of bacteria belonging to the beta-subclass of Proteobacteria and an overestimation of bacteria belonging to the gamma-subclass of Proteobacteria . Therefore, culture dependent enumerations of the gamma-subclass bacteria of the genus Acinetobacter in plants with enhanced biological phosphate removal (EBPR) resulted in significant overestimations. In situ identification by fluorescent oligonucleotide probing revealed that Acinetobacter numbers were below 8% of the active bacterial cells in the examined EBPR-plants. In situ hybridization techniques also bear the potential for the early and correct identification of filamentous bacteria as indicators for sludge bulking and foaming. A 16S ribosomal RNA targeted oligonucleotide probe specific for Sphaerotilus spec, was developed and successfully applied for in situ investigation of this filamentous bacterium.