In this study, methanogenic Archaea were isolated from five full-scale agricultural biogas plants (BGPs) located in Rhineland-Palatinate and Saarland, Germany, digesting maize silage and cattle manure. According to partial 16S rRNA gene sequences, the strains isolated from enrichment cultures were related to Methanoculleus bourgensis, Methanosarcina mazei, Methanosaeta concilii, and Methanobacterium formicicum. The 16S rRNA gene libraries of two representative BGPs screened with the direct amplified rDNA restriction analysis approach also revealed these Archaea to be present. Comparative phylogenetic analyses of reference strains and the isolates of genus Methanobacterium based on 16S and 23S rRNA gene sequences suggest two major groups of isolates, with both of them closely associated with Methanobacterium formicicum strain MFT. The affiliation of Methanobacterium isolates is further supported by denaturating gradient gel electrophoresis of 16S rRNA gene amplificates, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and specifically amplified polymorphic DNA-PCR (SAPD-PCR), a novel fingerprint approach applied to methanogenic Archaea for the first time. Signature sequence 03Mbf derived from the application of SAPD-PCR was subsequently used to develop a PCR-based primer system for the detection of Methanobacterium formicicum-related isolates and the reference strain in BGP samples. Amplification of 03Mbf fragments down to a minimal titer of 10(3) cells of Methanobacterium formicicum-related isolate Mb9 was possible under BGP fermenter-comparable conditions.