As part of a large metagenomic project, a one million fosmid library was constructed from using DNA extracted from sludge obtained fromsampled in mesophilic anaerobic digester treating wastewater. This library was screened by high density filter DNA-DNA hybridization procedure using various 16S rDNA probes. While exploring the 16S rDNA diversity, we identified fosmid clones possibly constituting a novel bacterial lineage : WWE3. Sequence analysis of a WWE3 fosmid clone revealed a 16S rRNA sequence with unusual features. We thus we developed specific oligonucleotides for PCR detection, quantitative dot-blot hybridization and FISH analysis. WWE3 specific 16S rRNA rDNA genes libraries were constructed from two anaerobic digesters. Phylogenetic analysis were performed using ARB database and software package. Further analysis with PAUP using different evolutionary models were carried out to resolve WWE3 phylogenetic position.Phylogenetic analysis of almost 660 full-length sequences confirmed that WWE3 forms a distinct and monophyletic group deeply branching apart from all bacterial divisions. WWE3 bacteria were detected by PCR in 20 out of 50 analyzed digesters from Europe and America. FISH analysis revealed that these organisms are oval-shaped and located inside sludge flocs. Due to their unusual 16S rRNA sequences, WWE3 sequences could not be retrieved by the conventional "16S" approach and there is only one affiliated sequence in the databases. Moreover, they represent a non negligiblesignificant fraction of the bacterial population in the studied anaerobic digesters. Sequence analysis of WWE3 fosmid clones will give us a first glimpse at the genomic properties of a member of this novel bacterial candidate division.