The phylogenetic diversity of Bacteria and Archaea within a biodegraded, mesothermic petroleum reservoir in the Schrader Bluff Formation of Alaska was examined by two culture-independent methods based on fosmid and small-subunit rRNA gene PCR clone libraries. Despite the exclusion of certain groups by each method, there was overall no significant qualitative difference in the diversity of phylotypes recovered by the two methods. The resident Bacteria belonged to at least 14 phylum-level lineages, including the polyphyletic Firmicutes, which accounted for 36.2% of all small-subunit rRNA gene-containing (SSU(+)) fosmid clones identified. Members of uncultured divisions were also numerous and made up 35.2% of the SSU(+) fosmid clones. Clones from domain Archaea accounted for about half of all SSU(+) fosmids, suggesting that their cell numbers were comparable to those of the Bacteria in this microbial community. In contrast to the Bacteria, however, nearly all archaeal clones recovered by both methods were related to methanogens, especially acetoclastic methanogens, while the plurality of bacterial fosmid clones was affiliated with Synergistes-like acetogenic Firmicutes that possibly degrade longer-chain carboxylic acid components in the crude oil to acetate. These data suggest that acetate may be a key intermediary metabolite in this subsurface anaerobic food chain, which leads to methane production as the primary terminal electron sink.