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A target-primedin siturolling circle amplification (in situRCA) protocol was developed for detection of single-copy genes inside bacterial cells and optimized withPseudomonas stutzeri,targeting nitrite and nitrous oxide reductase genes (nirSandnosZ). Two padlock probes were designed per gene to target both DNA strands; the target DNA was cut by a restriction endonuclease close to the probe binding sites, which subsequently were made accessible by 5′-3′ exonucleolysis. After hybridization, the padlock probe was circularized by ligation and served as template forin situRCA, primed by the probe target site. Finally, the RCA product inside the cells was detected by standard fluorescencein situhybridization (FISH). The optimized protocol showed high specificity and signal-to-noise ratio but low detection frequency (up to 15% for single-copy genes and up to 43% for the multi-copy 16S rRNA gene). Nevertheless, multiple genes (nirSandnosZ; nirSand the 16S rRNA gene) could be detected simultaneously inP. stutzeri.Environmental application ofin situRCA-FISH was demonstrated on activated sludge by the differential detection of two types ofnirS-defined denitrifiers; one of them was identified asCandidatusAccumulibacter phosphatis by combiningin situRCA-FISH with 16S rRNA-targeted FISH. While not suitable for quantification because of its low detection frequency,in situRCA-FISH will allow to link metabolic potential with 16S rRNA (gene)-based identification of single microbial cells.