ABSTRACT A toluene-degrading methanogenic consortium enriched from creosote-contaminated aquifer material was maintained on toluene as the sole carbon and energy source for 10 years. The species in the consortium were characterized by using a molecular approach. Total genomic DNA was isolated, and 16S rRNA genes were amplified by using PCR performed with kingdom-specific primers that were specific for 16S rRNA genes from either members of the kingdom Bacteria or members of the kingdom Archaea . A total of 90 eubacterial clones and 75 archaeal clones were grouped by performing a restriction fragment length polymorphism (RFLP) analysis. Six eubacterial sequences and two archaeal sequences were found in the greatest abundance (in six or more clones) based on the RFLP analysis. The relative abundance of each putative species was estimated by using fluorescent in situ hybridization (FISH), and the presence of putative species was determined qualitatively by performing slot blot hybridization with consortium DNA. Both archaeal species and two of the six eubacterial species were detected in the DNA and FISH hybridization experiments. A phylogenetic analysis of these four dominant organisms suggested that the two archaeal species are related to the genera Methanosaeta and Methanospirillum . One of the eubacterial species is related to the genus Desulfotomaculum , while the other is not related to any previously described genus. By elimination, we propose that the last organism probably initiates the attack on toluene.