The prokaryotic community composition of activated sludge from a seawater-processing wastewater treatment plant (Almeria, Spain) was investigated by using the rRNA approach, combining different molecular techniques such as denaturing gradient gel electrophoresis (DGGE), clone libraries and in situ hybridization (FISH and CARD-FISH). Most of the sequences retrieved in the DGGE and the clone libraries were similar to uncultured members of different phyla. The most abundant sequence recovered from Bacteria in the clone library corresponded to a bacterium from the Deinococcus-Thermus cluster (almost 77% of the clones), and the library included members from other groups such as the Alpha, Gamma and Delta subclasses of Proteobacteria, the Bacteroidetes and Firmicutes. Concerning the archaeal clone library, we basically found sequences related to different orders of methanogenic Archaea, in correspondence with the recovered DGGE bands. Enumeration of DAPI (4',6-diamidino-2-phenylindole) stained cells from two different activated sludge samples after a mechanical flocculation disruption revealed a mean cell count of 1.6×109ml-1. Around 94% of DAPI counts (mean value from both samples) hybridized with a Bacteria specific probe. Alphaproteobacteria were the dominant bacterial group (36% of DAPI counts), while Beta-, Delta- and Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes contributed to lower proportions (between 0.5-5.7% of DAPI counts). Archaea accounted only for 6% of DAPI counts. In addition, specific primers for amplification of the amoA (ammonia monooxygenase) gene were used to detect the presence of Beta, Gamma and archaeal nitrifiers, yielding positive amplifications only for Betaproteobacteria. This, together with negative in situ hybridizations with probes for well-known nitrifiying bacteria, suggests that nitrification is performed by still undetected microorganisms. In summary, the combination of the three approaches provided different and complementary pictures of the real assemblage composition and allowed to get closer to the main microorganisms involved in key processes of seawater-processing activated sludge. © 2011 The Authors. Journal compilation © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.